In vitro methods for processing lignin and other aromatic compounds

ABSTRACT

Enzymes for depolymerizing lignin. The enzymes include dehydrogenases, β-etherases, and glutathione lyases. The dehydrogenases can comprise one or more or LigD, LigO, LigN, and LigL. The β-etherases can comprise one or more of LigE, LigF, LigP, and BaeA. The glutathione lyases can comprise any one or more of LigG and a number of non-stereospecific, optionally recombinant glutathione lyases derived from  Sphingobium  sp. SYK-6,  Novosphingobium aromaticivorans, Escherichia coli, Streptococcus sanguinis, Phanerochaete chrysosporium , and other microorganisms. The enzymes can be combined in compositions and/or used in methods of processing lignin or other aromatic compounds in vitro.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under DE-FC02-07ER64494and DE-SC0018409 awarded by the US Department of Energy. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to the enzymatic depolymerization of lignin andthe enzymatic processing of other aromatic compounds.

BACKGROUND

Lignin provides structural rigidity in terrestrial plants and is largelycomprised of guaiacyl and syringyl monoaromatic phenylpropanoid unitsthat are covalently linked together in a purely chemical radicalcoupling polymerization process. The most prevalent type of inter-unitlinkage between units is the β-ether linkage in so-called β-ether unitsmaking up the lignin polymer.

Because lignin is rich in aromatics, lignin can potentially serve as asource for a number of valuable aromatic polymers, oligomers, andmonomers. However, lignin is notoriously difficult to process ordepolymerize into simpler compounds.

A number of chemical methods for depolymerizing lignin are known, butthese methods tend to involve high temperatures or pressures, expensivecatalysts, and organic solvents. Tools and methods of depolymerizinglignin that avoid at least some of these drawbacks are needed.

SUMMARY OF THE INVENTION

The invention at least in part is directed to an enzymatic system thatcatalyzes β-ether cleavage of actual lignin in vitro with the recyclingof cosubstrates NAD⁺ and GSH. In an exemplary version, the system usesthe known LigD, LigN, LigE, and LigF enzymes from Sphingobium sp. strainSYK-6, plus a novel, non-stereospecific glutathione transferase fromNovosphingobium aromaticivorans DSM12444 (NaGST_(Nu)). BaeA can be usedin addition to or in place of LigE. A glutathione reductase fromAllochromatium vinosum DSM180 (AvGR) is used to recycle thecosubstrates. The depolymerization of actual lignin with these enzymesis illustrated in FIGS. 1 and 16. The enzymatic depolymerization oflignin provided herein has several advantages over chemical routes as it(1) does not require high temperatures or pressures; (2) does notrequire expensive catalysts; (3) could be performed in an aqueousenvironment, eliminating the need for solvents (and subsequentseparation/recycle); and (4) results in a well-defined set of aromaticmonomers that have not undergone chemical transformations, and hence aremore amenable for downstream processing (i.e., upgrading).

More generally, the invention encompasses methods of processing lignin.One method of the invention comprises contacting lignin comprising β-O-4ether (β-ether) linkages in vitro with a dehydrogenase, a β-etherase,and a glutathione lyase. The dehydrogenase preferably comprises at leastone of LigD, LigO, LigN, and LigL. The β-etherase preferably comprisesat least one of LigE, LigF, LigP, and BaeA. The glutathione lyasepreferably comprises at least one of LigG and a non-stereospecificglutathione lyase comprising an amino acid sequence at least about 80%,85%, 90%, or 95% identical to any of: SEQ ID NO:18 (NaGST_(Nu));residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu)); SEQ ID NO:22(SYK6GST_(Nu)); residues 21-324 of SEQ ID NO:24 (recombinantSYK6GST_(Nu)); SEQ ID NO:26 (ecYghU); residues 21-313 of SEQ ID NO:28(recombinant ecYghU); SEQ ID NO:30 (ecYfcG); SEQ ID NO:32 (ssYghU); SEQID NO:34 (GST3); and SEQ ID NO:36 (PcUre2pB1).

In some versions, the non-stereospecific glutathione lyase comprises atleast one, at least two, at least three, at least four, at least five,at least six, at least seven, at least eight, at least nine, at leastten, or all of: asparagine or a conservative variant of asparagine at aposition corresponding to position 25 of SEQ ID NO:18 (NaGST_(Nu));threonine or a conservative variant of threonine at a positioncorresponding to position 51 of SEQ ID NO:18 (NaGST_(Nu)); asparagine ora conservative variant of asparagine at a position corresponding toposition 53 of SEQ ID NO:18 (NaGST_(Nu)); glutamine or a conservativevariant of glutamine at a position corresponding to position 86 of SEQID NO:18 (NaGST_(Nu)); lysine, a conservative variant of lysine,arginine, or a conservative variant of arginine at a positioncorresponding to position 99 of SEQ ID NO:18 (NaGST_(Nu)); isoleucine ora conservative variant of isoleucine at a position corresponding toposition 100 of SEQ ID NO:18 (NaGST_(Nu)); glutamate or a conservativevariant of glutamate at a position corresponding to position 116 of SEQID NO:18 (NaGST_(Nu)); serine, threonine, a conservative variant ofserine, or a conservative variant of threonine at a positioncorresponding to position 117 of SEQ ID NO:18 (NaGST_(Nu)); tyrosine ora conservative variant of tyrosine at a position corresponding toposition 166 of SEQ ID NO:18 (NaGST_(Nu)); arginine or a conservativevariant of arginine at a position corresponding to position 177 of SEQID NO:18 (NaGST_(Nu)); and tyrosine or a conservative variant oftyrosine at a position corresponding to position 224 of SEQ ID NO:18(NaGST_(Nu)).

In some versions, the contacting occurs in the presence of a glutathione(GSH) reductase that catalyzes reduction of glutathione disulfide(GSSG). The GSH reductase in some versions comprises an amino acidsequence at least 95% identical to SEQ ID NO:38 (AvGR).

Another method of the invention is a method of chemical conversion. Thechemicals converted in the method preferably comprise aromaticchemicals. One method comprises contacting a first compound in vitrowith a non-stereospecific glutathione lyase to yield a second compound.The non-stereospecific glutathione lyase may comprise any of thosedescribed above or elsewhere herein but preferably comprises anon-stereospecific glutathione lyase having an amino acid sequence atleast about 80%, 85%, 90%, or 95% identical to any of: SEQ ID NO:18(NaGST_(Nu)); residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));SEQ ID NO:22 (SYK6GST_(Nu)); residues 21-324 of SEQ ID NO:24(recombinant SYK6GST_(Nu)); SEQ ID NO:26 (ecYghU); residues 21-313 ofSEQ ID NO:28 (recombinant ecYghU); SEQ ID NO:30 (ecYfcG); SEQ ID NO:32(ssYghU); SEQ ID NO:34 (GST3); and SEQ ID NO:36 (PcUre2pB1). The firstcompound preferably has a structure of Formula I or a salt thereof:

wherein R¹, R², and R³ are each independently —H, —OH, —O-alkyl,—O-lignin, or -lignin; R⁴ is —H, —OH, —SH, —COOH, —SO₃H, or —O-lignin;and SG is glutathione bound in an S or R configuration. The secondcompound has a structure of Formula II or a salt thereof:

wherein R¹, R², R³, and R⁴ are as defined above.

The invention also encompasses compositions. The compositions mayinclude any of the components employed in the methods described herein.One composition of the invention comprises lignin comprising β-O-4 etherlinkages, a dehydrogenase, a β-etherase, and a glutathione lyase. Thedehydrogenase, a β-etherase, and a glutathione lyase preferably includeany of those described above or elsewhere herein. In some versions, thecomposition further comprises a glutathione (GSH) reductase thatcatalyzes reduction of glutathione disulfide (GSSG). The GSH reductasein some versions comprises a sequence at least about 95% identical toSEQ ID NO:38 (AvGR).

The invention also encompasses recombinant enzymes. The recombinantenzymes may include recombinant versions of any of the enzymes describedabove or elsewhere herein. The recombinant enzymes preferably include arecombinant non-stereospecific glutathione lyase. The non-stereospecificglutathione lyase may comprise any of those described above or elsewhereherein but preferably comprises a non-stereospecific glutathione lyasehaving an amino acid sequence at least about 80%, 85%, 90%, or 95%identical to any of: SEQ ID NO:18 (NaGST_(Nu)); residues 21-313 of SEQID NO:20 (recombinant NaGST_(Nu)); SEQ ID NO:22 (SYK6GST_(Nu)); residues21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu)); SEQ ID NO:26(ecYghU); residues 21-313 of SEQ ID NO:28 (recombinant ecYghU); SEQ IDNO:30 (ecYfcG); SEQ ID NO:32 (ssYghU); SEQ ID NO:34 (GST3); and SEQ IDNO:36 (PcUre2pB1). The recombinant non-stereospecific glutathione lyasepreferably comprises at least one non-native modification selected fromthe group consisting of an amino acid addition, an amino acid deletion,and an amino acid substitution.

The invention also encompasses processed lignin or compounds obtainedthrough any of the methods described herein.

The objects and advantages of the invention will appear more fully fromthe following detailed description of the preferred embodiment of theinvention made in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the sphingomonadales β-etherase pathway, used to break theβ-aryl ether ((3-O-4) bond of compounds such asguaiacylglycerol-β-guaiacyl ether (GGE). Names of enzymes catalyzingeach reaction are taken from Sphingobium sp. SYK-6 (LigLNDOFEPG; Masaiet al. 2003, Sato et al. 2009; Tanamura et al. 2011), Novosphingobiumsp. MBESO4 (GST3) (Ohta et al. 2015), or Novosphingobium aromaticivorans(NaGST_(Nu)) (Example 1); the color of each enzyme name matches thearrow color of the reaction it catalyzes. Below each enzyme is listedwhich of the three sphingomonads investigated in Example 1(Novosphingobium sp. PP1Y, Novosphingobium aromaticivorans, andSphingobium xenophagum) are predicted to contain that enzyme. The α, β,and γ carbons of GGE are labeled in the topmost molecule, and thestereochemical designations of all chiral molecules are shown. As shown,metabolism of GGE begins with the stereoselective, NAD⁺-dependentoxidation (by LigD, LigO, LigL, LigN) of the β-aryl alcohol into theα-ketone, β-(2-methoxyphenoxy)-γ-hydroxypropiovanillone (MPHPV) (Sato etal. 2009). β(S)- and β(R)-MPHPV are then cleaved by stereospecificglutathione (GSH)-dependent β-etherases (e.g., LigF, LigE/P) to yieldthe β(R)- and β(S)-stereoisomers of the glutathione conjugateβ-glutathionyl-γ-hydroxypropiovanillone (GS-HPV) and guaiacol (Masai etal. 2003, Gall and Kim et al. 2014). GSH-dependent enzymes that removethe glutathione moiety from GS-HPV to form hydroxypropiovanillone (HPV)and glutathione disulfide (GSSG) have been identified in vitro: LigGreacts specifically with β(R)-GS-HPV (Masai et al. 2003), whereas GST3and NaGST_(Nu) react with both the β(R)- and β(S)-stereoisomers (Ohta etal. 2015).

FIGS. 2A and 2B show cell densities and extracellular metaboliteconcentrations of N. aromaticivorans cultures grown in SMB containing 3mM GGE (FIG. 2A, panels A-F), or 4 mM vanillate and 1.5 mM GGE (FIG. 2B,panels G-L). Data are shown for strains 12444Δ1879 (panels A,B,G,H),12444Δ2595 (panels C,D,I,J), and 12444ecyghU (panels E,F,K,L). They-axes of panels H, J, and L use different scales. For comparison, celldensity data for cultures grown in SMB containing 4 mM vanillate onlyare included in panels G, I, and K.

FIG. 3 shows growth and extracellular metabolite levels in arepresentative culture of Novosphingobium aromaticivorans 12444Δ1879 inSMB containing 200 μM GGE. The rate of GGE metabolism by N.aromaticivorans (˜200 μM within ˜30 h) is comparable to that ofErythrobacter sp. SG61-1L (˜200 μM within ˜60 h, though this strainapparently cannot further metabolize guaiacol; Palamuru et al. 2015) andNovosphingobium sp. MBESO4 (˜900 μM within ˜40 h, though this strainapparently cannot assimilate carbon from GGE into cell material; Ohta etal. 2015).

FIGS. 4A and 4B show cell densities and extracellular metaboliteconcentrations from cultures of Novosphingobium sp. PP1Y (FIG. 4A,panels A,B,E,F) and Sphingobium xenophagum (FIG. 4B, panels C,D,G,H)grown in SMB containing 3 mM GGE (FIGS. 4A and 4B, panels A,B,C,D); or 4mM vanillate (FIG. 4A, panels E,F) or glucose (FIG. 4B, panels G,H) with1.5 mM GGE. The y-axis segments of panels D,F,H of FIGS. 4A and 4B areat different concentration scales. For comparison, cell density data forcultures grown in SMB containing 4 mM vanillate only are included inpanels.

FIG. 5 shows an amino acid sequence alignment of various exemplarynon-stereospecific glutathione lyases of the invention. The alignedglutathione lyase sequences include those of NaGST_(Nu) (SEQ ID NO:18),SYK6GST_(Nu) (SEQ ID NO:22), ecYghU (SEQ ID NO:26), ecYfcG (SEQ IDNO:30), ssYghU (SEQ ID NO:32), GST3 (SEQ ID NO:34), and PcUre2pB1 (SEQID NO:36). The NaGST_(Nu), ecYghU, ssYghU, ecYfcG, and PcUre2pB1proteins have been structurally characterized. Residues fromstructurally solved proteins predicted to interact with GSH or GSSGmolecules indicated with “*” and correspond to the following residues inSEQ ID NO:18 (NaGST_(Nu)): Asn25, Thr51, Asn53, Gln86, Lys99, Ile100,Glu116, Ser117, and Arg177. Residues predicted to be involved in thereaction mechanism are indicated with “#” and correspond to thefollowing residues in SEQ ID NO:18 (NaGST_(Nu)): Tyr166, Tyr224, andPhe288. Alignment was made using MAFFT version 7 (mafft.cbrc.jp) (Katohet al. 2002) in MegAlign Pro, which is part of the Lasergene 14.0 suite(DNASTAR, Madison, Wis.).

FIG. 6 shows kinetics of the conversion of the β(R)- (A) and β(S)- (B)stereoisomers of GS-HPV into HPV. Reactions used 8 nM NaGST_(Nu), 195 nMecYghU, 195 nM ecYfcG, or 47 (A) or 18 (B) nM SYK6-GST_(Nu). The linesare non-linear least squares best fits to the experimental data usingthe Michaelis-Menten equation.

FIG. 7 shows time courses for the reaction of cell extracts from N.aromaticivorans strains 12444Δ1879 (A) and 12444Δ2595 (B) with a racemicsample of β(R)- and β(S)-MPHPV. The red dotted line in panel B indicatesthe time at which recombinant NaGST_(Nu) and additional GSH were addedto the reaction.

FIG. 8 shows the structure of NaGST_(Nu) (pdb 5uuo). (A) Domainstructure of one subunit of the homodimer. The GST1 N-terminal(thioredoxin) domain extends from Val39 to Gly129 (green), the GST2C-terminal domain extends from Ser135 to Leu257 (maroon), and anextension of the C-terminal extends from Val258 to Phe288 (orange). (B)Residue contacts to the GSH dithiol (60% occupancy) and GS-SG (40%) inthe NaGST_(Nu) 5uuo structure. The carbon atoms of GSH1 are coloredlight cyan; those of GSH2 are dark cyan; those of GS-SG are lightorange. NaGST_(Nu) residues with 3.2 Å or shorter contacts to eitherGSH1 or GSH2 are labeled, and colored according to domain origin definedabove. Selected distances between interacting atoms are shown.

FIG. 9 shows a comparison of the active site in closely related Nu-classGSTs. (A) Alignment of subunits of NaGST_(Nu) (pdb 5uuo; open form,white; closed form, blue), ecYghU (pdb 3ec8; orange), and ssYghU (pdb4mzw; green). (B) Spatially conserved positions (Phe82, Tyr224 andLys262) in the open form subunit of NaGST_(Nu) that define a triangleover the entrance to the active site used to approximate the size of thechannel opening. (C) Positions (Phe82, Tyr224 and Phe288) and triangledefined in the closed form of NaGST_(Nu). (D) Positions (Gly83, Tyr225and Gly263) and triangle defined in ecYghU. (E) Positions (Met80, Tyr222and Ala255) and triangle defined in ssYghU.

FIG. 10 shows: (A) Molecular docking and energy-minimized positions of(R)- and (S)-GS-HPV (orange and purple lines, respectively) in theoutwardly branching entrance to the active site of NaGST_(Nu) (thesulfur atom of GSH1 in the active site is visible as a yellow sphere);(B) Predicted residue interactions with (R)-GS-HPV; and (C) Predictedresidue interactions with (S)-GS-HPV.

FIG. 11 shows a proposed mechanism for NaGST_(Nu)-catalyzed cleavage ofeither (R)- (left column) or (S)- (right column) thioether bonds inGS-HPV. (A) SG1 is close to the thiol of GHS2. Conserved Thr51, whichlies within 3.0 Å of SG1, provides a hydrogen bond that promotes attackof SG1 on SG2 and formation of G1S-SG2. (B) Rupture of the thioetherbond is facilitated by Y166, which stabilizes a transient enolateintermediate. (C) Collapse of the enolate to the observed products.

FIG. 12 shows modeling of substrates into the active sites of NaGST_(Nu)and ecYghU. Panels (A) and (B) show modeling of β(R)- and β(S)-GS-HPVinto NaGST_(Nu). Panels (C) and (D) show modeling of β(R)- andβ(S)-GS-HPV into ecYghU. Panels (E) and (F) show modeling of β(R)- andβ(S)-GS-conjugated syringyl phenylpropanoids into NaGST_(Nu). Coloringfor NaGST_(Nu) is the same as in FIG. 8 (with residues for ecYghU inparentheses): E4 (T5) to P38 (P39) in gray; V39 (V40) to G129 (G130) ingreen; V130 (Y131) to T134 (Q135) in gray; S135 (D136) to L257 (1257) inmaroon; V258 (V258) to F288 (G288) in orange. Residues predicted to beinvolved in catalysis of the glutathione lyase reaction are Tyr66 andTyr224 (Tyr167 and Tyr225 in ecYghU). Resides that contribute todifferences in active site channel interiors between NaGST_(Nu) andecYghU are Phe82 and Phe288 in NaGST_(Nu), and Arg260 and Asn262 inecYghU. Carbon atoms of GSH1 are yellow, and those of the GS-conjugatedsubstrates (GS-HPV or the syringyl analogue) are cyan.

FIG. 13 shows a phylogenetic analysis of Nu-classglutathione-S-transferases. BLAST searches of the NCBI non-redundantprotein database were performed using NaGST_(Nu) and GST3 as queries.The top 5,000 hits from both of these searches were collected; everyfifth member from each of these sets was transferred into a new combinedset of 2,000 proteins. Sequences for SYK6GST_(Nu) and ecYfcG were addedto the combined set, to give a set of 2,002 proteins. Proteins in thisset were aligned using MAFFT in MegAlign Pro, which is part of theLasergene 14.0 suite (DNASTAR, Madison, Wis.). A phylogenetic tree wascalculated via the maximum likelihood method in RAxML v8.2.3(Stamatakis, 2014), using 100 rapid bootstrap inferences. The tree wasvisualized using Interactive Tree of Life v3 (http://itol.embl.de).Enzymes experimentally reported here (NaGST_(Nu), SYK6GST_(Nu), ecYghU,ecYfcG) or elsewhere (GST3 (Ohta et al., 2015)) to be able to convertGS-HPV into HPV are identified.

FIG. 14 shows percent methanol in the running buffer during HPLCanalysis as used in the experiments in Example 1. The remainder of therunning buffer was Buffer A (5 mM formic acid, 5% acetonitrile in H₂O),and the flow rate was 1 mL/minute.

FIG. 15 shows absorbance (280 nm) and retention times of metabolitesidentified by HPLC as described in Example 1.

FIG. 16 shows aromatic monomers and the β-etherase pathway. Panel (A)shows the structures of predominant monomeric phenylpropanoids found inlignin, guaiacyl (G, in blue), syringyl (S, in red), as well as tricin(T, in green) units. Arrows indicate where inter-unit linkages areformed during radical coupling reactions. Dashed lines indicatepositions that may form additional covalent bonds during post-couplingreaction mechanisms. Panel (B) shows β-etherase pathway-mediateddegradation of the diaromatic β-ether-linked model compound GGE viaNAD⁺-dependent dehydrogenases LigD and LigN to form GGE-ketone (alsoreferred to herein as “β-(2-methoxyphenoxy)-γ-hydroxypropiovanillone” or“MPHPV”) and NADH. GGE-ketone undergoes GSH-dependent β-ether cleavageby β-etherase enzymes LigE and LigF to yield guaiacol and GS-HPV asmonoaromatic derivative products. GS-HPV undergoes GSH-dependentthioether cleavage by NaGST_(Nu) or LigG, producing GSSG andmonoaromatic product HPV. As indicated by the dashed arrows, AvGRrecycles co-substrates GSH and NAD⁺ via NADH-dependent reduction ofGSSG. For reactions involving an R- or S-configured epimer as thesubstrate, the isomer towards which each enzyme exhibits activity isshown in grey text. Panel (C) shows how β-etherase pathway enzymesdegrade GTE through intermediate GTE-ketone to yield tricin and HPV.

FIGS. 17A and 17B show HPLC chromatographic traces of substrates andproducts of β-etherase pathway assays using NAD⁺ (2.0 mM), GSH (4.0 mM),and erythro-GGE (6.0 mM). Elution time of compounds (absorbance at 280nm) are highlighted by shading: NAD⁺ and NADH (˜3.0 min), GS-HPV (˜4.5min), HPV (˜9.0 min), guaiacol (˜14.0 min), erythro-GGE (˜18.0 min),threo-GGE (˜19.0 min), and GGE-ketone (˜21.5 min). Structures of GS-HPV,HPV, guaiacol, GGE, and GGE-ketone are shown in FIG. 16 (B). Panel (A)of FIG. 17A shows a control sample to which no enzymes were added. After4 h incubation with one of the following combinations of enzymaticcatalysts (50 μg/mL each): the remaining panels in FIGS. 17A and 17Bshow products in assays containing (FIG. 17A, panel B) LigDNEF andNaGST_(Nu); (FIG. 17A, panel C) LigDNEF, NaGST_(Nu), and AvGR; (FIG.17B, panel D) LigDNEF and LigG; and (FIG. 17B, panel E) LigDNEF, LigG,and AvGR.

FIG. 18 shows time-dependent changes in concentrations of erythro-GGE (

), threo-GGE (

), GGE-ketone (-o-), HPV (

), and guaiacol (

) in an assay that, at 0 min, was supplemented with NAD⁺ (2.0 mM), GSH(4.0 mM), and erythro-GGE (6.0 mM), as well as (50 μg/mL each) LigD,LigN, LigE, LigF, NaGST_(Nu), and AvGR. Numbers in parentheses representthe measured concentration (mM) of each compound after 4 h ofincubation. Structures of HPV, guaiacol, GGE, and GGE-ketone are shownin FIG. 16 (B). erythro-GGE is a mixture of enantiomers (αR,βS)-GGE and(αS,βR)-GGE. threo-GGE is a mixture of enantiomers (αS,βS)-GGE and(αR,βR)-GGE.

FIGS. 19A and 19B show HPLC chromatographic traces of β-etherase pathwayproducts in reactions containing the indicated enzymes and NAD⁺ (5.0mM), GSH (5.0 mM), and GTE (1.0 mM, a 6:1 mixture of erythro-GTE tothreo-GTE). Elution time of compounds (absorbance at 280 nm) arehighlighted: NAD⁺ and NADH (˜3.0 min), GS-HPV (˜5.5 min), HPV (˜16.5min), tricin (˜51.5 min), threo-GTE (˜52.0 min), erythro-GTE (˜56.0min), and GTE-ketone (˜58.5 min). Panel (A) of FIG. 19A shows thecontrol sample to which no enzymes were added. After 4 h incubation withone of the following combinations of enzymatic catalysts (50 μg/mLeach): the panels in FIG. 19B show products in assays containing (panelB) LigD, LigN, LigE, LigF, and NaGST_(Nu); (panel C) LigD, LigN, LigEand LigF; (panel D) LigD and LigN. Structures of GS-HPV, HPV, tricin,GTE, and GTE-ketone are shown in FIG. 16 (C).

FIG. 20 shows analytical GPC traces (λ=200 nm) showing the sizedistribution of (A) unfractionated HP lignin (MW=8,665), (B) fractionsof MCS lignin collected from preparative GPC, and (C) the fractions thatwere pooled and used as the substrate in enzyme assays: fraction 1(MW=11,550), fraction 2 (MW=10,780), and fraction 3 (MW=9,340). Forreference, the approximate MW of a 10-mer is indicated with a dashedline.

FIG. 21 shows HPLC chromatographic traces of coupled β-etherase pathwayreactions supplemented with NAD⁺ (2.0 mM), GSH (4.0 mM), and HP ligninfractions (2.2 mg mL⁻¹). Elution times of compounds (absorbance at 280nm) are highlighted: NAD⁺ and NADH (˜3.0 min), HPS (˜10.0 min), unknown(grey, ˜20.0 min), and syringaresinol (˜21.5 min). Panel (A) shows thepooled GPC fractions 1 (MW=11,550), 2 (MW=10,780), and 3 (MW=9,340)without enzyme addition. Panel (B) shows after 4 h incubation with LigD,LigN, LigE, LigF, NaGST_(Nu) and AvGR (50 μg/mL each) and pooled HPlignin fractions 1-3 as the substrate.

FIG. 22 shows analytical GPC traces (λ=200 nm) showing the distributionof (A) unfractionated MCS lignin (MW=5,980), (B) fractions of MCS lignincollected from preparative GPC, and (C) the fractions used as substratesin enzyme assays: fraction 1 (MW=10,710), fraction 5 (MW=5,370),fraction 8 (MW=1,390), fraction 14 (MW=660), and fraction 17 (MW=460).For reference, the approximate MW of a 10-mer is indicated with a dashedline.

FIGS. 23A and 23B show HPLC chromatographic traces of β-etherase pathwayenzyme activities in reactions containing NAD⁺ (2.0 mM), GSH (4.0 mM),and MCS lignin or the indicated MCS lignin fractions (2.2 mg mL⁻¹).Elution times (absorbance at 280 nm) are highlighted by shading: NAD⁺(˜3.0 min), HPV (˜9.0 min), HPS (˜10.0 min), unknowns (grey, ˜18.0-19.0min), and tricin (˜26.5 min), and an unknown broad peak (orange,˜22.0-29.0 min). Panel A in FIG. 23A shows the control sample(unfractionated by GPC) to which no enzymes were added. The remainingpanels in FIGS. 23A and 23B show products after 4 h incubation with 50μg/mL each of LigD, LigN, LigE, LigF, NaGST_(Nu) and AvGR, and one ofthe following MCS lignin fractions: (FIG. 23A, panel B) fraction 1(MW=10,710), (FIG. 23A, panel C) fraction 5 (MW=5,3670), (FIG. 23B,panel D) fraction 8 (MW=1,390), (FIG. 23B, panel E) fraction 14(MW=660), and (FIG. 23B, panel F) fraction 17 (MW=460). Structures ofHPV, HPS, and tricin are shown in FIG. 16.

FIG. 24 shows the extracellular concentration of metabolites with growthof N. aromaticivorans strains 12444Δ1879 (effective wild-type; A),12444ΔligE (B), 12444Δ2872 (C), 12444ΔligEΔ2872 (D), and 12444ΔligEΔ2873(E) in Standard Mineral Base (SMB) containing 3 mM vanillate and 1 mMerythro-GGE. Extracellular concentrations of vanillate are not shown.The segments of panels A-E use different x-axis scales for clarity ofpresentation.

FIG. 25 shows stereoisomer(s) of MPHPV remaining in the media in samplesfrom the final time point of the experiment shown for FIG. 24 for the12444ΔligEΔ2872 and 12444ΔligEΔ2873 cultures (F-H and I-K,respectively). The final time point samples were combined with H₂O,recombinant LigF, or recombinant LigE to determine which stereoisomer(s)of MPHPV remained in the media. The two stereoisomers each oferythro-GGE, threo-GGE, MPHPV, and GS-HPV are not distinguishable in ourmethod of analysis.

FIG. 26 shows reactions of racemic MPHPV with the Saro_2872 andSaro_2873 polypeptides individually and combined. Racemic (β(R) andβ(S)) MPHPV was initially mixed with H₂O (A), or the Saro_2872 orSaro_2873 polypeptides individually (B,C), or combined (D). The samplescontaining both Saro_2872 and Saro_2873 was then split and combined witheither LigE (Saro_2405) or LigF1 (Saro_2091). All reactions contain atleast 5 mM GSH. The β(R) and β(S) stereoisomers of both MPHPV and GS-HPVare indistinguishable in our analysis.

FIG. 27 shows gel permeation chromatography of various BaeA (Saro_2872and Saro_2873 heterodimer) variants and other standard proteins of knownmolecular weights.

FIG. 28 shows a phylogentic tree of LigE and LigF homologues and theSaro_2872 and Saro_2873 polypeptides.

FIG. 29 shows a polypeptide sequence alignment of Saro_2872 andSaro_2873 with known LigF enzymes.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention includes methods of processing lignin. Theterm “lignin” used herein refers to any compound comprising covalentlylinked phenylpropanoid units. Phenylpropanoids include compoundscommonly referred to as “phenylpropane units,” “lignin monomer units,”or variants thereof, and are well-known compounds in the art.Phenylpropanoids include a substituted or non-substituted six-carbonaromatic phenyl group and a substituted or non-substituted three-carbontail. The phenyl group and tail may be substituted or unsubstituted. Thetail may be saturated or unsaturated. The substitutions on the phenylgroup may include hydroxy and alkoxy (e.g., methoxy) groups, amongothers. The substitutions on the tail may include hydroxy, alkoxy,carboxy, thiol, and sulfonate groups, among others. Exemplaryphenylpropanoid units include the p-hydroxyphenyl (H), guaiacyl (G), andsyringyl (S) units derived from p-coumaryl alcohol, coniferyl alcohol,and sinapyl alcohol, respectively. The phenylpropanoid units can belinked to other phenylpropanoid units, flavonoid units such as tricin,or other types of chemical units. The phenylpropanoid units arepreferably linked through radical coupling. Exemplary linkages includeβ-O-4, 5-5, β-5, β-1, 4-O-5, and β-β linkages. See, e.g., Santos et al.2013.

Two types of lignin include natural lignin and synthetic lignin.“Natural lignin” refers to lignin in which the phenylpropanoid units arecovalently linked in nature (in vivo), regardless of whether or not thelignin is subsequently processed in vitro. Natural lignin encompasseslignin from non-genetically engineered plants as well as geneticallyengineered plants. The genetically engineered plants include plants thathave been genetically engineered for altered lignin production, such asincorporation of ferulates moieties (U.S. Pat. Nos. 8,569,465 and9,388,285), flavan-3-ols and/or gallic acid derivatives (U.S. Pat. No.8,685,672), high syringyl content (see the examples), or othermodifications (U.S. Pat. Nos. 9,487,794; 9,441,235; and 9,493,783).“Synthetic lignin” refers to lignin in which the phenylpropanoid unitsare covalently linked in vitro. Methods of covalently linkingphenylpropanoid units in vitro through radical coupling reactions arewell known in the art. See, e.g., Grabber et al. 1996.

“Processing” or grammatical variants thereof refers herein to modifyinglignin in any manner to result in at least one structural change.Processing can occur through chemical, physical, or enzymatic methods.Examples of processing include depolymerization, oxidation, acidtreatment, base treatment, enzyme treatment, heating, mechanicalshearing, etc. The processing may depolymerize the lignin (at least tosome degree), chemically modify the lignin, physically break apart thelignin, remove or add functional groups on the lignin, or result inother structural changes.

Certain methods of the invention are directed to enzymaticallyprocessing lignin comprising β-O-4 linkages (β-ether linkage) to breakat least a portion of the β-O-4 linkages and/or release compounds fromthe lignin. The processing can advantageously be performed in vitrousing one or more enzymes. The term “in vitro” in this context refers toprocessing with enzymes in which the enzymes are not actively producedby any intact, living organisms, and is contrasted with in vivoprocessing, in which the enzymes involved with the processing areactively produced by one or more intact, living organisms. Thus, in someversions, the enzymes involved in the processing are not activelyproduced during the duration of the processing. In some versions, theprocessing occurs in the absence of intact, living microorganisms. Insome versions, the processing occurs in the absence of any intact,living Sphingobium species, Erythrobacter species, Novosphingobiumspecies, Escherichia species, Streptococcus species, and/orPhanerochaete species.

In some versions, the enzymes involved in the processing are purifiedenzymes. The term “isolated” or “purified” means a material that isremoved from its original environment, for example, the naturalenvironment if it is naturally occurring, or a fermentation broth if itis produced in a recombinant host cell fermentation medium. A materialis said to be “purified” when it is present in a particular compositionin a higher or lower concentration than the concentration that existsprior to the purification step(s). For example, with respect to acomposition normally found in a naturally-occurring or wild typeorganism, such a composition is “purified” when the final compositiondoes not include some material from the original matrix. As anotherexample, where a composition is found in combination with othercomponents in a recombinant host cell fermentation medium, thatcomposition is purified when the fermentation medium is treated in a wayto remove some component of the fermentation, for example, cell debrisor other fermentation products, through, for example, centrifugation ordistillation. As another example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated, whether suchprocess is through genetic engineering or mechanical separation. Inanother example, a polynucleotide or protein is said to be purified ifit gives rise to essentially one band in an electrophoretic gel or ablot.

A first step in the enzymatic processing involves contacting lignincomprising β-O-4 (β-ether) linkages with a dehydrogenase. Thedehydrogenase is preferably capable of oxidizing α-hydroxyls on β-etherunits to corresponding α-ketones. The term “β-ether unit” is used hereinto refer to a phenylpropanoid moiety linked to a second phenylpropanoidmoiety via a β-O-4 linkage. See, e.g., FIGS. 1 and 16. Exemplary enzymesused for this step include any one or more of LigD, LigO, LigN, andLigL. Exemplary LigD, LigO, LigN, and LigL enzymes include those fromSphingobium sp. SYK-6 having the sequences of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, and SEQ ID NO:8, respectively, which are encoded by SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, respectively. LigD,LigO, LigN, and LigL enzymes are found in organisms other thanSphingobium sp. SYK-6 and can be used in place of or in addition tothose from Sphingobium sp. SYK-6. Modified forms of the native LigD,LigO, LigN, and LigL enzymes can also suitably be used, provided themodified forms maintain the activity of the native enzymes. Suchmodified forms that maintain the activity of the native enzymes are alsoreferred to herein as LigD, LigO, LigN, and LigL enzymes. The modifiedforms comprise sequences at least 95% identical to the amino acidsequences of the corresponding native enzymes.

The LigD and LigO enzymes from Sphingobium sp. SYK-6 have a specificityfor α-hydroxyls in the α(R) stereochemical configuration. The LigN andLigL enzymes from Sphingobium sp. SYK-6 have a specificity forα-hydroxyls in the α(S) stereochemical configuration. To ensureefficient processing in a non-stereospecific manner, it is preferredthat the lignin is contacted with at least one α(R) stereospecificenzyme and least one α(S) stereospecific enzyme. Accordingly, preferredcombinations include one or more of LigD and LigO with one or more ofLigN and LigL.

LigD, LigO, LigN, and LigL homologs from Erythrobacter sp. SG61-1L canreact with all four possible stereoisomers of GGE (see Palamuru et al.2015) and can be used in place of or in combination with the enzymesfrom Sphingobium sp. SYK-6.

A second step in the enzymatic processing involves contactingpreprocessed (preliminarily processed) lignin, such as a product of thefirst step, with a β-etherase. The β-etherase is preferably capable ofcatalyzing glutathione-dependent β-ether cleavage to yield aβ-glutathione-linked phenylpropanoid unit in place of the β-etherphenylpropanoid unit. See, e.g., FIGS. 1 and 16. The second step can beperformed simultaneously with or subsequent to the first step. Exemplaryenzymes used for this step include any one or more of LigE, LigF, LigP,and BaeA.

Exemplary LigE, LigF, and LigP enzymes include those from Sphingobiumsp. SYK-6 having the sequences of SEQ ID NO:10, SEQ ID NO:12, and SEQ IDNO:14, respectively, which are encoded by SEQ ID NO:9, SEQ ID NO:11, andSEQ ID NO:13, respectively. LigE, LigF, and LigP enzymes are found inorganisms other than Sphingobium sp. SYK-6 and can suitably be used inplace of or in addition to those from Sphingobium sp. SYK-6. Modifiedforms of the native LigE, LigF, and LigP enzymes can also suitably beused, provided the modified forms maintain the activity of the nativeenzymes. Such modified forms that maintain the activity of the nativeenzymes are also referred to herein as LigE, LigF, and LigP enzymes. Themodified forms comprise sequences at least 95% identical to the aminoacid sequences of the corresponding native enzymes.

“BaeA” refers to a heterodimer of a first polypeptide having an aminoacid sequence of SEQ ID NO:40 or an amino acid sequence at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical thereto and asecond polypeptide having an amino acid sequence of SEQ ID NO:42 or anamino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, ormore identical thereto. An exemplary BaeA is a heterodimer of Saro_2872from Novosphingobium aromaticivorans (encoded by SEQ ID NO:39 andrepresented by SEQ ID NO:40) and Saro_2873 from Novosphingobiumaromaticivorans (encoded by SEQ ID NO:41 and represented by SEQ IDNO:42). The second polypeptide preferably comprises an asparagine or aconservative variant of asparagine at a position corresponding toposition 14 of SEQ ID NO:41 and/or a serine or a conservative variant ofserine at a position corresponding to position 15 of SEQ ID NO:42.

The LigE and LigP enzymes from Sphingobium sp. SYK-6 and BaeA have aspecificity for cleaving β-ether linkages in the β(R) stereochemicalconfiguration. The LigF enzyme from Sphingobium sp. SYK-6 has aspecificity for cleaving β-ether linkages in the β(S) stereochemicalconfiguration. To ensure efficient processing in a non-stereospecificmanner, it is preferred that the substrate is contacted with at leastone β(R) stereospecific enzyme and least one β(S) stereospecific enzyme.Accordingly, preferred combinations include one or more of LigE, LigP,and BaeA with LigF.

LigE/P and LigF homologues from other organisms are also expected to bestereospecific. See for example Gall and Ralph et al. 2014. SLG_08660(SYK-6 LigE), PP1Y_AT11664 (Novosphingobium sp. PP1Y), SLG_32600 (SYK-6LigP), and Saro_2405 (Novosphingobium aromaticivorans) were all shown tobe specific for β(R)-compounds. Likewise, SLG_08650 (SYK-6 LigF),Saro_2091 (Novosphingobium aromaticivorans), and Saro_2865(Novosphingobium aromaticivorans) were all shown to be specific forβ(S)-compounds.

A third step in the enzymatic processing involves contactingpreprocessed lignin, such as a product of the second step, with aglutathione lyase. The glutathione lyase is preferably capable ofcleaving glutathione from β-glutathione-linked phenylpropanoid units.The cleavage may use glutathione as a cosubstrate and produceglutathione disulfide in the process. See, e.g., FIGS. 1 and 16. Thethird step can be performed simultaneously with or subsequent to thesecond step and/or the first and second steps. Exemplary enzymes usedfor this step include any one or more of LigG from Sphingobium sp. SYK-6having the amino acid sequence of SEQ ID NO:16 (encoded by SEQ IDNO:15), a Nu-class glutathione 5-transferase (GST) from Novosphingobiumaromaticivorans DSM 12444 having the amino acid sequence of SEQ ID NO:18(NaGST_(Nu)) (encoded by SEQ ID NO:17), a recombinant NaGST_(Nu) havingthe amino acid sequence of residues 21-313 of SEQ ID NO:20 (encoded bySEQ ID NO:19 prior to cleavage), a Nu-class GST from Sphingobium sp.SYK-6 having the amino acid sequence of SEQ ID NO:22 (SYK6GST_(Nu))(encoded by SEQ ID NO:21), a recombinant SYK6GST_(Nu) having the aminoacid sequence of residues 21-324 of SEQ ID NO:24 (encoded by SEQ IDNO:23 prior to cleavage), a Nu-class GST from Escherichia coli DH5αhaving the amino acid sequence of SEQ ID NO:26 (ecYghU) (encoded by SEQID NO:25), a recombinant ecYghU having the amino acid sequence ofresidues 21-313 of SEQ ID NO:28 (encoded by SEQ ID NO:27 prior tocleavage), a Nu-class GST from Escherichia coli DH5α having the aminoacid sequence of SEQ ID NO:30 (ecYfcG) (encoded by SEQ ID NO:29), aNu-class GST from Streptococcus sanguinis SK36 having the amino acidsequence of SEQ ID NO:32 (ssYghU) (encoded by SEQ ID NO:31), a Nu-classGST from Novosphingobium sp. MBESO4 having the amino acid sequence ofSEQ ID NO:34 (GST3) (encoded by SEQ ID NO:33), and a Nu-class GST fromPhanerochaete chrysosporium RP-78 having the amino acid sequence of SEQID NO:36 (PcUre2pB1) (encoded by SEQ ID NO:35).

The LigG from Sphingobium sp. SYK-6 has a specificity forβ-glutathione-linked phenylpropanoid units with the glutathione moietieslinked in the β(R) stereochemical configuration and is referred toherein as a “stereospecific glutathione lyase.” By contrast, theNu-class glutathione S-transferase (GST) from Novosphingobiumaromaticivorans DSM 12444 having the amino acid sequence of SEQ ID NO:18(NaGST_(Nu)) (encoded by SEQ ID NO:17), the recombinant NaGST_(Nu)having the amino acid sequence of residues 21-313 of SEQ ID NO:20(encoded by SEQ ID NO:19 prior to cleavage), the Nu-class GST fromSphingobium sp. SYK-6 having the amino acid sequence of SEQ ID NO:22(SYK6GST_(Nu)) (encoded by SEQ ID NO:21), the recombinant SYK6GST_(Nu)having the amino acid sequence of residues 21-324 of SEQ ID NO:24(encoded by SEQ ID NO:23 prior to cleavage), the Nu-class GST fromEscherichia coli DH5α having the amino acid sequence of SEQ ID NO:26(ecYghU) (encoded by SEQ ID NO:25), the recombinant ecYghU having theamino acid sequence of residues 21-313 of SEQ ID NO:28 (encoded by SEQID NO:27 prior to cleavage), the Nu-class GST from Escherichia coli DH5αhaving the amino acid sequence of SEQ ID NO:30 (ecYfcG) (encoded by SEQID NO:29), the Nu-class GST from Streptococcus sanguinis SK36 having theamino acid sequence of SEQ ID NO:32 (ssYghU) (encoded by SEQ ID NO:31),the Nu-class GST from Novosphingobium sp. MBESO4 having the amino acidsequence of SEQ ID NO:34 (GST3) (encoded by SEQ ID NO:33), and theNu-class GST from Phanerochaete chrysosporium having the amino acidsequence of SEQ ID NO:36 (PcUre2pB1) (encoded by SEQ ID NO:35) arecapable or predicted to be capable of cleaving β-glutathione-linkedphenylpropanoid units with the glutathione moieties linked in either theβ(R) or β(S) stereochemical configuration and are referred to herein as“non-stereospecific glutathione lyases.” To ensure efficient processingin a non-stereospecific manner, it is preferred that the substrate iscontacted with at least one non-stereospecific glutathione lyase.Accordingly, preferred combinations include one or more of any of thenon-stereospecific glutathione lyases described herein with or withoutLigG.

Other non-stereospecific glutathione lyases that can be used in place ofor in addition to the non-stereospecific glutathione lyases describedabove include enzymes at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, or at least about 95% identical to anyone of SEQ ID NO:18 (NaGST_(Nu)), residues 21-313 of SEQ ID NO:20(recombinant NaGST_(Nu)), SEQ ID NO:22 (SYK6GST_(Nu)), residues 21-324of SEQ ID NO:24 (recombinant SYK6GST_(Nu)), SEQ ID NO:26 (ecYghU),residues 21-313 of SEQ ID NO:28 (recombinant ecYghU), SEQ ID NO:30(ecYfcG), SEQ ID NO:34 (GST3); SEQ ID NO:32 (ssYghU), and SEQ ID NO:36(PcUre2pB1). Such enzymes may be native enzymes or modified forms ofnative enzymes.

As discussed in the following examples, a number of residues of thenon-stereospecific glutathione lyases described herein play at leastsome role in the enzymatic activity. See, e.g., FIG. 5. These includeAsn25 of SEQ ID NO:18 (NaGST_(Nu)), Thr51 of SEQ ID NO:18 (NaGST_(Nu)),Asn53 of SEQ ID NO:18 (NaGST_(Nu)), Gln86 of SEQ ID NO:18 (NaGST_(Nu)),Lys99 of SEQ ID NO:18 (NaGST_(Nu)), Ile100 of SEQ ID NO:18 (NaGST_(Nu)),Glu 116 of SEQ ID NO:18 (NaGST_(Nu)), Ser117 of SEQ ID NO:18(NaGST_(Nu)), Tyr166 of SEQ ID NO:18 (NaGST_(Nu)), Arg177 of SEQ IDNO:18 (NaGST_(Nu)), Tyr224 of SEQ ID NO:18 (NaGST_(Nu)), andcorresponding residues in the other enzymes. A number of these residuesare conserved across all the non-stereospecific glutathione lyasesdescribed herein. These include Thr51 of SEQ ID NO:18 (NaGST_(Nu)),Asn53 of SEQ ID NO:18 (NaGST_(Nu)), Gln86 of SEQ ID NO:18 (NaGST_(Nu)),Ile100 of SEQ ID NO:18 (NaGST_(Nu)), Glu 116 of SEQ ID NO:18(NaGST_(Nu)), Arg177 of SEQ ID NO:18 (NaGST_(Nu)), and correspondingresidues in the other enzymes.

Accordingly, suitable enzymes at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, or at least about 95% identical to thenon-stereospecific glutathione lyases described herein preferablycomprise at least one, at least two, at least three, at least four, atleast five, at least six, at least seven, or all of threonine or aconservative variant of threonine at a position corresponding toposition 51 of SEQ ID NO:18 (NaGST_(Nu)), asparagine or a conservativevariant of asparagine at a position corresponding to position 53 of SEQID NO:18 (NaGST_(Nu)), glutamine or a conservative variant of glutamineat a position corresponding to position 86 of SEQ ID NO:18 (NaGST_(Nu)),lysine, a conservative variant of lysine, arginine, or a conservativevariant of arginine at a position corresponding to position 99 of SEQ IDNO:18 (NaGST_(Nu)), isoleucine or a conservative variant of isoleucineat a position corresponding to position 100 of SEQ ID NO:18(NaGST_(Nu)), glutamate or a conservative variant of glutamate at aposition corresponding to position 116 of SEQ ID NO:18 (NaGST_(Nu)),serine, threonine, a conservative variant of serine, or a conservativevariant of threonine at a position corresponding to position 117 of SEQID NO:18 (NaGST_(Nu)), arginine or a conservative variant of arginine ata position corresponding to position 177 of SEQ ID NO:18 (NaGST_(Nu)).

Suitable enzymes at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, or at least about 95% identical to thenon-stereospecific glutathione lyases described herein more preferablycomprise at least one, at least two, at least three, at least four, atleast five, at least six, at least seven, at least eight, at least nine,at least ten, or all of asparagine or a conservative variant ofasparagine at a position corresponding to position 25 of SEQ ID NO:18(NaGST_(Nu)), threonine or a conservative variant of threonine at aposition corresponding to position 51 of SEQ ID NO:18 (NaGST_(Nu)),asparagine or a conservative variant of asparagine at a positioncorresponding to position 53 of SEQ ID NO:18 (NaGST_(Nu)), glutamine ora conservative variant of glutamine at a position corresponding toposition 86 of SEQ ID NO:18 (NaGST_(Nu)), lysine, a conservative variantof lysine, arginine, or a conservative variant of arginine at a positioncorresponding to position 99 of SEQ ID NO:18 (NaGST_(Nu)), isoleucine ora conservative variant of isoleucine at a position corresponding toposition 100 of SEQ ID NO:18 (NaGST_(Nu)), glutamate or a conservativevariant of glutamate at a position corresponding to position 116 of SEQID NO:18 (NaGST_(Nu)), serine, threonine, a conservative variant ofserine, or a conservative variant of threonine at a positioncorresponding to position 117 of SEQ ID NO:18 (NaGST_(Nu)), tyrosine ora conservative variant of tyrosine at a position corresponding toposition 166 of SEQ ID NO:18 (NaGST_(Nu)), arginine or a conservativevariant of arginine at a position corresponding to position 177 of SEQID NO:18 (NaGST_(Nu)), and tyrosine or a conservative variant oftyrosine at a position corresponding to position 224 of SEQ ID NO:18(NaGST_(Nu)).

Positions in a given enzyme that correspond to positions in a givensequence such as SEQ ID NO:18 (NaGST_(Nu)), SEQ ID NO:40 (Saro_2872),and SEQ ID NO:42 (Saro_2873), or any other sequence provided herein, canbe identified through alignment of the sequence of the enzyme with thegiven sequence. A number of sequence alignment algorithms are known inthe art. Exemplary sequence alignment algorithms include MAFFT version 7(mafft.cbrc.jp) (Katoh et al. 2002) and Clustal W and other Clustalprograms (Larkin et al. 2007). Other algorithms and programs are knownin the art.

“Conservative variant” refers to residues that are functionally similarto a given residue such that one or more of the functionally similarresidues may substitute for the given residue. Conservative variants ofthe standard amino acids are well known in the art. In some versions,aliphatic, non-polar amino acids (Gly, Ala, Ile, Leu, and Val) areconservative variants of one another. In some versions, aliphatic, polaramino acids (Cys, Ser, Thr, Met, Asn, Tyr and Gln) are conservativevariants of one another. In some versions, aromatic amino acids (Phe,Tyr, Trp, and His) are conservative variants of one another. In someversions, basic amino acids (Lys, Arg, and His) are conservativevariants of one another. In some versions, acidic amino acids (Asp andGlu) are conservative variants of one another. In some versions, anamino acid with an acidic side chain, Glu or Asp, is a conservativevariant of its uncharged counterpart, Gln or Asn, respectively; or viceversa. In some versions, each of the following groups contains otherexemplary amino acids that are conservative variants of one another: Alaand Gly; Asp and Glu; Asn and Gln; Arg and Lys; Ile, Leu, Met, and Val;Phe, Tyr, and Trp; Ser and Thr; and Cys and Met.

A fourth step in the enzymatic processing involves conducting the firstthree steps in the presence of a glutathione (GSH) reductase thatcatalyzes reduction of glutathione disulfide (GSSG). The GSH reductaseregenerates the GSH cosubstrate of the second and third steps from theGSSG co-product of the third step and regenerates the NAD⁺ cosubstrateof the first step from the NADH co-product of the first step. See, e.g.,FIGS. 1 and 16. An exemplary GSH reductase is the GSH reductase fromAllochromatium vinosum DSM180 (AvGR) having the sequence of SEQ ID NO:38(encoded by SEQ ID NO:37). Other GSH reductases are known in the art andcan be used in this step. Modified forms of the native AvGR and othersuitable enzymes can also suitably be used, provided the modified formsmaintain the activity of the native enzymes. The modified forms comprisesequences at least 95% identical to the amino acid sequences of thecorresponding native enzymes.

The enzymatic processing outlined above is capable of depolymerizinglignin and/or releasing compounds therefrom. Compounds that are capableof being released from lignin include monomeric phenylpropanoid unitsand monomeric flavones. Examples of monomeric phenylpropanoid unitscapable of being released from lignin include monomeric guaiacylphenylpropanoid units, monomeric syringyl phenylpropanoid units, andmonomeric p-hydroxyphenyl phenylpropanoid units. Examples of flavonescapable of being released from lignin include monomeric tricin units.

The lignin subjected to the enzymatic processing outlined above may haveany of a number of average molecular weights (MW). The average molecularweight (MW) in some versions, for example, may be at least about 100, atleast about 200, at least about 300, at least about 400, at least about500, at least about 600, at least about 700, at least about 800, atleast about 900, at least about 1000, at least about 1,250, at leastabout 1,500, at least about 1,750, at least about 2,000, at least about2,500, at least about 3,000, at least about 3,500, at least about 4,000,at least about 4,500, at least about 5,000, at least about 6,000, atleast about 7,000, at least about 8,000, at least about 9,000, at leastabout 10,000, at least about 11,000, at least about 13,000, at leastabout 15,000, or more. The average molecular weight (MW) in someversions may be up to about 150, up to about 200, up to about 300, up toabout 400, up to about 500, up to about 600, up to about 700, up toabout 800, up to about 900, up to about 1000, up to about 1,250, up toabout 1,500, up to about 1,750, up to about 2,000, up to about 2,500, upto about 3,000, up to about 3,500, up to about 4,000, up to about 4,500,up to about 5,000, up to about 6,000, up to about 7,000, up to about8,000, up to about 9,000, up to about 10,000, up to about 11,000, up toabout 13,000, up to about 15,000, up to about 20,000 or more.

Certain methods of the invention are directed to methods of chemicalconversion. Such methods may comprise contacting a first compound invitro with a non-stereospecific glutathione lyase to yield a secondcompound.

The non-stereospecific glutathione lyase used in the methods of chemicalconversion may comprise any of the non-stereospecific glutathione lyasesdescribed herein. Preferred non-stereospecific glutathione lyasesinclude non-stereospecific glutathione lyases comprising an amino acidsequence at least about 60% identical, at least about 65% identical, atleast about 70% identical, at least about 75% identical, at least about80% identical, at least about 85% identical, at least about 90%identical, or at least about 95% identical to any of SEQ ID NO:18(NaGST_(Nu)), residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu)),SEQ ID NO:22 (SYK6GST_(Nu)), residues 21-324 of SEQ ID NO:24(recombinant SYK6GST_(Nu)), SEQ ID NO:26 (ecYghU), residues 21-313 ofSEQ ID NO:28 (recombinant ecYghU), SEQ ID NO:30 (ecYfcG), SEQ ID NO:32(ssYghU), SEQ ID NO:34 (GST3), and SEQ ID NO:36 (PcUre2pB1).

The first compound contacted with the non-stereospecific glutathionelyase in the methods of chemical conversion preferably has a structureof Formula I or a salt thereof:

wherein: R¹, R², and R³ are each independently —H, —OH, —O-alkyl,—O-lignin, or -lignin; R⁴ is —H, —OH, —SH, —COOH, —SO₃H, or —O-lignin;and SG is glutathione bound in an S or R configuration.

The second compound yielded by contacting the first compound with thenon-stereospecific glutathione lyase in the methods of chemicalconversion preferably has a structure of Formula II or a salt thereof:

wherein R¹, R², R³, and R⁴ are as defined above.

Contacting the first compound with the non-stereospecific glutathionelyase in the methods of chemical conversion may occur in in the presenceof NADH and a GSH reductase that catalyzes reduction of glutathionedisulfide, such as a GSH reductase comprising an amino acid sequence atleast 95% identical to SEQ ID NO:38 (AvGR).

The first compound may be generated by contacting lignin comprisingβ-O-4 ether linkages with any dehydrogenase described herein and/or anyβ-etherase described herein. The lignin may be contacted with theseenzymes in vitro.

Another aspect of the invention includes recombinant enzymes. Therecombinant enzymes may be used in any of the methods described herein.The recombinant enzymes may comprise recombinant versions of any enzymedescribed or encompassed herein, including non-stereospecificglutathione lyases or other enzymes. The term “recombinant” used withreference to an enzyme refers to non-naturally occurring enzymescontaining two or more linked polypeptide sequences. Thus, therecombinant enzymes may contain one or more non-native modificationsselected from the group consisting of an amino acid addition, an aminoacid deletion, and an amino acid substitution. “Non-native modification”refers to a modification that is not found in any native protein. Therecombinant enzymes can be produced by recombination methods,particularly genetic engineering techniques, or can be produced bychemical synthesis.

In some versions, the recombinant enzyme comprises a recombinantnon-stereospecific glutathione lyase of the invention. The recombinantnon-stereospecific glutathione lyase may be a recombinant version of anyof the non-stereospecific glutathione lyases described or encompassedherein. Preferred recombinant non-stereospecific glutathione lyasesinclude non-stereospecific glutathione lyases comprising an amino acidsequence at least about 60% identical, at least about 65% identical, atleast about 70% identical, at least about 75% identical, at least about80% identical, at least about 85% identical, at least about 90%identical, or at least about 95% identical to any of: SEQ ID NO:18(NaGST_(Nu)); residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));SEQ ID NO:22 (SYK6GST_(Nu)); residues 21-324 of SEQ ID NO:24(recombinant SYK6GST_(Nu)); SEQ ID NO:26 (ecYghU); residues 21-313 ofSEQ ID NO:28 (recombinant ecYghU); SEQ ID NO:30 (ecYfcG); SEQ ID NO:32(ssYghU); SEQ ID NO:34 (GST3); and SEQ ID NO:36 (PcUre2pB1). The designof amino acid deletions and substitutions in the recombinantnon-stereospecific glutathione lyases can be guided by the alignment ofthe native non-stereospecific glutathione lyases provided in FIG. 5.

Amino acid additions in the recombinant enzymes of the invention,including the recombinant non-stereospecific glutathione lyases, maycomprise the addition of any amino acid on the N-terminus, theC-terminus, or both the N-terminus and C-terminus of any native enzyme.

The added amino acids may comprise fusion tags. A number of fusion tagsare known in the art. Some fusion tags are used for protein detection.These include green fluorescent protein (GFP) and its many variants(Tsien 1998). Some fusion tags are used for increasing expression andsolubility of proteins. These include maltose binding protein (MBP),small ubiquitin-like modifier (SUMO), and glutathione S-transferase(GST), among others (Bell et al. 2013, Butt et al. 2005). Some fusiontags, sometimes referred to as “affinity tags,” are used forpurification, detection with antibodies, or other uses. A number ofaffinity tags are known in the art. Exemplary affinity tags include theHis tag, the Strep II tag, the T7 tag, the FLAG tag, the S tag, the HAtag, the c-Myc tag, the dihydrofolate reductase (DHFR) tag, the chitinbinding domain tag, the calmodulin binding domain tag, and the cellulosebinding domain tag. The sequences of each of these tags are well-knownin the art.

The recombinant enzymes of the invention, including the recombinantnon-stereospecific glutathione lyases, may comprise a peptide cleavagesequence. The peptide cleavage sequence is preferably disposed betweenthe enzyme portion and any fusion tag attached thereto. This permitsseparation of the fusion tag from the target protein, which may beuseful for certain applications. The peptide cleavage sequence may be arecognition sequence for a site-specific peptidase. A number ofsite-specific peptidases are known in the art. These include Arg-Cproteinase, Asp-N endopeptidase, Asp-N endopeptidase+N-terminal glu,BNPS-Skatole, caspase1, caspase2, caspase3, caspase4, caspase5,caspase6, caspase7, caspase8, caspase9, caspase10, chymotrypsin-highspecificity (C-term to [FYW], not before P), chymotrypsin-lowspecificity (C-term to [FYWML], not before P), clostripain(clostridiopeptidase B), CNBr, enterokinase, factor Xa, formic acid,glutamyl endopeptidase, granzymeB, hydroxylamine, iodosobenzoic acid,Lys-C, Lys-N, NTCB (2-nitro-5-thiocyanobenzoic acid), neutrophilelastase, pepsin (pH1.3), pepsin (pH>2), proline-endopeptidase,proteinase K, SUMO proteases (Ulp1, Senp2, and SUMOstar), staphylococcalpeptidase I, subtilisin BPN, tobacco etch virus (TEV) protease,thermolysin, thrombin, and trypsin, and variants thereof, among others.The cleavage recognition sites for these and other site-specificpeptidases are well known in the art. Exemplary peptide cleavagesequences include the ExxYxQ↓(G/S) recognition sequence of TEV (andAcTEV and ProTEV), the LVPR↓G recognition sequence of thrombin, theIEGR↓x recognition sequence of factor Xa, and the DDDDK↓x recognitionsequence of enterokinase.

The elements and method steps described herein can be used in anycombination whether explicitly described or not.

All combinations of method steps as used herein can be performed in anyorder, unless otherwise specified or clearly implied to the contrary bythe context in which the referenced combination is made.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the content clearly dictates otherwise.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, from 5to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All patents, patent publications, and peer-reviewed publications (i.e.,“references”) cited herein are expressly incorporated by reference tothe same extent as if each individual reference were specifically andindividually indicated as being incorporated by reference. In case ofconflict between the present disclosure and the incorporated references,the present disclosure controls.

It is understood that the invention is not confined to the particularconstruction and arrangement of parts herein illustrated and described,but embraces such modified forms thereof as come within the scope of theclaims.

Example 1 Nu-Class Glutathione-S-Transferases can Act as GlutathioneLyases in the Bacterial Pathway for Breaking β-Aryl Ether Bonds inLignin Summary

As a major component of plant cells walls, lignin is a potentialrenewable source of valuable chemicals. Several sphingomonad bacteriahave been identified that can use glutathione to break the β-aryl etherbond commonly found between phenylpropanoid units of the ligninheteropolymer. To explore bacterial strategies for depolymerizinglignin, we tested the abilities of three sphingomonads to metabolize theβ-aryl ether containing dimeric aromatic compoundguaiacylglycerol-β-guaiacyl ether (GGE). We found that Novo-sphingobiumaromaticivorans metabolized GGE at amongst the fastest rates thus farreported. After the β-aryl ether bond of GGE is broken, the glutathionemoiety must be removed from the resultant phenylpropanoid conjugate(α-glutathionyl-β-hydroxypropio-vanillone (GS-HPV)). We found that aNu-class glutathione-S-transferase (GST) is necessary and sufficient forremoving glutathione from both the R and S stereoisomers of GS-HPV in N.aromaticivorans. To investigate the prevalence of this glutathione lyaseactivity within related proteins, we tested Nu-class GSTs fromSphingobium sp. SYK-6 and Escherichia coli and found that they alsocleave both stereoisomers of GS-HPV. We solved the crystal structure ofthe N. aromaticivorans Nu-class GST and used it to develop models forhow this enzyme binds and cleaves both stereoisomers of GS-HPV.

Significance

There is considerable interest is identifying biological strategies toproduce valuable products from renewable resources. The non-edible,lignocellulosic, fraction of plant biomass has been identified as arenewable source of bio-based products, but the properties of thearomatic polymer lignin present a major hurdle in realizing this goal.Here, we describe a novel role for a Nu-class glutathione-S-transferase(GST) in cleavage of the β-aryl ether bond that connects many aromaticunits in lignin. We show that homologues of this enzyme from otherbacteria also have this activity, suggesting that this function may becommon throughout this widespread class of enzymes. Structural andbiochemical analysis of Nu-class GSTs are used to model substratebinding and cleavage by these enzymes.

Introduction

As society looks to diversify its sources of fuels and chemicals, thereare compelling reasons to develop renewable sources for them.Lignocellulosic plant biomass is the most abundant renewable material onEarth, so it is a promising but underutilized feedstock for generatingproducts currently derived from petroleum or other non-renewablesources. Lignin, which can compose ˜25% of plant biomass (U.S. DOE2015), is a phenylpropanoid heteropolymer containing several classes ofcovalent linkages, including a majority of β-aryl ether (β-O-4) bonds(Adler 1977). The properties of lignin create challenges to using it asan industrial raw material. Because of its abundance and potentialvalue, there is interest in developing economical and environmentallysustainable methods to depolymerize lignin to its aromatic substituents.We are interested in analyzing biological processes for lignindepolymerization and conversion to valuable chemicals.

The β-etherase pathway of sphingomonadales bacteria, which cleaves theβ-aryl ether bond (FIG. 1), is a promising route for lignindepolymerization. Although several species are known to contain thispathway (Masai et al. 1989, Palamuru et al. 2015, Ohta et al. 2015),characterizing the strategies and proteins employed could help todevelop biological systems for depolymerizing lignin. In this work, wetested sphingomonads predicted to contain the β-etherase pathway (Ohtaet al. 2015), and found that N. aromaticivorans most rapidly andcompletely metabolized the dimeric aromatic compoundguaiacylglycerol-β-guaiacyl ether (GGE; FIG. 1). We also found that theSaro_2595 gene of N. aromaticivorans encodes a previouslyuncharacterized Nu-class glutathione-S-transferase (named hereNaGST_(Nu)) that is capable of cleaving glutathione from both the β(R)-and β(S)-stereoisomers of the pathway intermediateβ-glutathionyl-γ-hydroxypropiovanillone (GS-HPV; FIG. 1) in vitro, andwe show that it is the sole enzyme in N. aromaticivorans required forthis reaction in vivo.

Nu-class GSTs are found in many organisms (Stourman et al. 2011;Mashiyama et al., 2014), but their physiological roles are unknown.Although the Escherichia coli Nu-class GSTs ecYfcG and ecYghU can reducethe disulfide bond of 2-hydroxyethyl disulfide in vitro (Stourman et al.2011, Wadington et al. 2009, Wadington et al. 2010), the physiologicalrelevance of this reaction has not been established. To test theprevalence of the glutathione lyase activity in Nu-class GSTs, weassayed ecYghU, ecYfcG, and a Nu-class GST from Sphingobium sp. SYK-6(encoded by SLG_04120; named here SYK6GST_(Nu)), and found that theyalso cleave β(R)- and β(S)-GS-HPV in vitro. Furthermore, ecYghUcomplements growth of an N. aromaticivorans ΔNaGST_(Nu) mutant, showingthat it is active in vivo.

Crystal structures reported here show that NaGST_(Nu) is similar toecYghU (Stourman et al. 2011) and Streptococcus sanguinis SK36 YghU(Patskovsky et al.), although there are notable differences in thechannels leading to the active sites. We propose a mechanism for theglutathione lyase activity of the Nu-class GSTs and use molecularmodeling to show how the active site channels of these enzymes canaccommodate the β(R)- and β(S)-stereoisomers of GS-HPV. Indeed, theapproach to the active site of these Nu-class GSTs can accommodate avariety of GSH-conjugated substrates, independent of C—S bondstereochemistry, including other GSH-conjugates derived from lignindepolymerization, or ones that might be found in organisms that do notmetabolize lignin.

General Materials and Methods

Bacterial Strains and Growth Media.

Strains used are listed in Table 1. Unless otherwise noted, E. colicultures were grown in Lysogeny Broth (LB), and shaken at ˜200 rpm at37° C. For routine storage and manipulation, sphingomonad cultures weregrown in LB at 30° C. GluSis is a modification of Sistrom's minimalmedium (Sistrom 1962) in which the succinate has been replaced by 22.6mM glucose. Standard Mineral Base (SMB) (Stanier et al. 1966) contains20 mM Na₂HPO₄, 20 mM KH₂PO₄, 1 g/L (NH₄)₂SO₄, and 20 mL Hutner'svitamin-free concentrated base (adapted from (Cohen et al. 1957), butlacking nicotinic acid, thiamin, and biotin) per liter, final pH 6.8.SMB was supplemented with carbon sources as described below. Whereneeded to select for plasmids, media were supplemented with 50 μg/mLkanamycin and/or 10 μg/mL chloramphenicol.

TABLE 1 Bacterial strains and plasmids used. Strains Relevantcharacteristics References Novosphingobium aromaticivorans strains DSM12444 Wild-type; also called F199 Fredrickson et al. 1991 12444Δ1879 DSM12444 ΔSaro_1879 This study 12444Δ2595 12444Δ1879 ΔSaro_2595 This study12444ecyghU 12444Δ2595 containing E. coli yghU at This study theSaro_2595 locus Novosphingobium sp. Wild-type Notomista et al. 2011 PP1YSphingobium Wild-type; also called BN6^(T) and DSM Stolz et al. 2000;Pal et xenophagum NBRC 6383^(T) al. 2006 107872 Escherichia coli strainsDH5α F-ϕ80lacZ ΔM15 Δ(lacZYA-argF) Bethesda Research U169 recA1 endA1hsdR17 (rK−, Laboratories mK+) phoA supE44 λ− thi-1 gyrA96 Taylor (1993)relA1 S17-1 recA pro hsdR RP4-2-Tc::Mu-Km::Tn7 Simon et al. 1983 TurboF′proA⁺B⁺ lacI^(q) ΔlacZM15/ New England Biolabs fhuA2 Δ(lac-proAB) glnVgalK16 galE15 R(zgb-210::Tn10)Tet^(S) endA1 thi-1 Δ(hsdS-mcrB)5 NEB5-alpha Competent fhuA2 Δ(argF-lacZ)U169 phoA glnV44 New England BiolabsE. coli ϕ80Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 E. cloni 10GF- mcrA Δ(mrr-hsdRMS- Lucigen mcrBC) endA1 recA1 ϕ 80d/acZΔM15ΔlacX74araD139 Δ(ara,leu)7697galU galK rpsL nupG λ- tonA (StrR) B834 F-hsdSmetE gal ompT Wood 1966; Doherty 1995 Plasmids pK18mobsacB pMB1ori sacBkan^(R) mobT oriT(RP4) Schafer et al. 1994 lacZα pK18msB-MCS1pK18mobsacB lacking the multiple Present example cloning site pVP302Klac promoter lacI, Tev site rtxA (V. Supplemental cholera) kan^(R);coding sequence for Materials of Gall and 8 × His-tag Ralph et al. 2014pRARE2 p15a ori camR; tRNA genes for 7 rare Novagen codons in E. colipK18msB/ΔSaro1879 pK18 mobsacB containing genomic Present exampleregions flanking Saro_1879 pK18msB/ΔSaro2595 pK18 mobsacB containinggenomic Present example regions flanking Saro_2595 pK18msB/ecyghU-pK18mobsacB containing E. coli yghU Present example Δ2595 between theSaro_2595 flanking regions pVP302K/Ctag-2595 pVP302K containingSaro_2595 Present example upstream of rtxA and His-tag sequencepVP302K/Untagged2595 pVP302K containing Saro_2595 Present examplepVP302K/Ntag-2595 pVP302K containing Saro_2595 Present exampledownstream of His-tag coding sequence and Tev protease sitepVP302K/Ntag-ecyghU pVP302K containing yghU downstream Present exampleof His-tag coding sequence and Tev protease site pVP302K/Ntag-ecyfcGpVP302K containing yfcG downstream Present example of His-tag codingsequence and Tev protease site pVP302K/Ntag- pVP302K containingSLG_04120 Present example SLG_04120 downstream of His-tag codingsequence and Tev protease site

Construction of Defined N. aromaticivorans Mutants.

We deleted Saro_1879 from the Novosphingobium aromaticivorans DSM 12444genome to create a strain (12444Δ1879) amenable to genomic modificationsusing a sacB-containing vector. We used 12444Δ1879 to generate strainsin which Saro_2595 was deleted from the genome (12444Δ2595) and in whichSaro_2595 was replaced in the genome by the E. coli yghU gene(12444ecyghU).

Sphingomonad Growth Experiments.

Cell densities were measured using a Klett-Summerson photoelectriccolorimeter with a red filter. For N. aromaticivorans, 1 Klett Unit (KU)is equal to ˜8×10⁶ cells/mL (Table 2). Experimental cultures of N.aromaticivorans and Novosphingobium sp. PP1Y were grown in SMBcontaining either vanillate or GGE alone (4 mM and 3 mM, respectively),or a combination of vanillate and GGE (4 mM and 1.5 mM, respectively).For S. xenophagum cultures, vanillate was replaced by glucose, since wefound this strain to be unable to metabolize vanillate. N.aromaticivorans was also grown in SMB containing 200 μM GGE. Startercultures were grown in SMB with 4 mM vanillate or glucose, and cellswere pelleted and washed with PBS (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mMNaCl, 2.7 mM KCl; pH=7.4). Pellets were resuspended into culture mediumand used to inoculate experimental cultures to initial cell densities of<5 KU.

Cultures were incubated at 30° C., in 125 mL conical flasks containing20-40 mL of medium and shaken at ˜200 rpm. Aliquots (400-600 μL) wereremoved at specified time points and filtered through 0.22 μm syringetip filters (e.g., Whatman Puradisc filters, GE Healthcare, Piscataway,N.J.) before HPLC analysis of extracellular aromatics. Every culture wasgrown at least three times; data shown are from representative cultures.

TABLE 2 Relationship between Klett Units (KU) and colony forming units(CFUs) for Novosphingobium aromaticivorans +/− sucrose (CFU mL⁻¹ KU⁻¹).−sucrose +sucrose DSM 12444 8.0 (±3.2) × 10⁶ 0 12444Δ1879 8.1 (±1.7) ×10⁶ 7.3 (±3.4) × 10⁶To acquire these data, cultures of Novosphingobium aromaticivorans DSM12444 and 12444Δ1879, were grown in liquid medium. Cell densities weremeasured using a Klett-Summerson photoelectric colorimeter with a redfilter. Cultures were diluted, then plated onto solid media +/−10%sucrose.

Enzyme Purifications.

Genes Saro_2595, E. coli yghU, E. coli yfcG, and SLG_04120 fromSphingobium sp. SYK-6 (codon-optimized for expression in E. coli) wereindividually cloned into plasmid pVP302K (Gall and Ralph et al. 2014) sothat transcripts from the plasmids would be translated into proteinscontaining a His₈-tag connected to the N-terminus via a tobacco etchvirus (TEV) protease recognition site. Recombinant proteins wereexpressed in E. coli B834 (Wood 1966, Doherty et al. 1995) containingplasmid pRARE2 (Novagen, Madison, Wis.) grown for ˜24 h at 27° C. inZYM-5052 Autoinduction Medium (Studier 2005) containing kanamycin andchloramphenicol. Recombinant proteins were purified essentially asdescribed previously (Gall and Kim et al. 2014); see below formodifications to the procedure. After removal of His₈-tags using TEVprotease, recombinant proteins retained a Ser-Ala-Ile-Ala-Gly-peptide ontheir N-termini, derived from the linker between the protein and the TEVprotease recognition site. Recombinant LigE and LigF1 from N.aromaticivorans were purified as previously described (Gall and Ralph etal. 2014).

Kinetics Analysis of GS-HPV Cleavage.

The Reaction Buffer (RB) consisted of 25 mM Tris-HCl (pH 8.5) and 25 mMNaCl. The β(R)- and β(S)-stereoisomers of GS-HPV were separatelygenerated by incubating racemic MPHPV (0.46 mM) in RB with 5 mM reducedglutathione (GSH) and either 38 μg/mL LigF1 or 36 μg/mL LigE,respectively, for several h. This sample, containing a single GS-HPVstereoisomer, guaiacol, and the unreacted MPHPV stereoisomer, wasdiluted with RB to achieve the desired concentration of GS-HPV for thetime course reaction (0.005, 0.011, 0.022, 0.096, or 0.193 mM). Anadditional 5 mM GSH (dissolved in RB) was added prior to initiation ofeach time course. At time zero, 100 μL of the indicated enzyme sample(resuspended in RB) was combined with 1800 μL of the diluted GS-HPVreaction mixture to achieve final concentrations of 8 nM NaGST_(Nu), 195nM ecYghU, 195 nM ecYfcG, or 47 nM (for β(R)-GS-HPV reactions) or 18 nM(for β(S)-GS-HPV reactions) SYK6GST_(Nu). Assays were performed at 25°C., and at specified time points, 300 μL of the reaction was removed andcombined with 100 μL 1 M HCl (Acros Organics; Geel, Belgium) to stop thereaction before HPLC analysis to quantify HPV formed.

N. aromaticivorans Crude Cell Extract Assays.

N. aromaticivorans cells were grown in 500 mL conical flasks containing267 mL SMB medium with 4 mM vanillate and 1 mM GGE. When cell densitiesreached ˜1×10⁹ cells/mL, cells were lysed by the sonication procedureused to generate E. coli lysates for protein purification. Samples werecentrifuged at 7000×g for 15 minutes, and the supernatants were used ascrude cell extracts.

Assays containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10 mM GSH, 0.407mM racemic MPHPV (a mixture of the β(R)- and β(S)-isomers) and cellularextract from 12444Δ1879 or 12444Δ2595 (final concentrations of 269 and186 μg protein/mL, respectively) were performed at 30° C. At definedtime points, 300 μL aliquots were combined with 100 μL 1 M HCl to stopthe reaction before HPLC analysis. At the indicated time, recombinantNaGST_(Nu) (30 μg protein/mL) was added to the 12444Δ2595 cell extractreaction, along with an additional 10 mM GSH.

HPLC Analysis.

After extracellular aromatics were identified using LC-MS, routineanalysis and quantification of aromatics were performed using an UltraAQ C18 5 μm column (Restek, Bellefonte, Pa.) attached to a System GoldHPLC (Beckman Coulter, Brea, Calif.) with running buffers andchromatography conditions described in FIG. 14. The eluent was analyzedfor light absorbance between 191 and 600 nm, and absorbances at 280 nmwere used for quantification of aromatic metabolites by comparing peakareas to those of standards (retention times of measured metabolites areshown in FIG. 15).

Production of cDNA Libraries from N. aromaticivorans Cultures andReal-Time PCR.

N. aromaticivorans cultures were grown in 120 mL of SMB containingeither 4 mM vanillate or 4 mM vanillate and 1 mM GGE. When the culturesreached densities of ˜8×10⁸ cells/mL, 40 mL were removed and combinedwith 5.71 mL ice-cold Stop Solution (95% ethanol, 5% acid phenol:chloroform (5:1 solution, pH 4.5)). These mixtures were centrifuged at4° C. for 12 min at 6,000×g. Cell pellets were resuspended into 2 mLLysis Solution (2% SDS, 16 mM EDTA in RNase-free water), then incubatedat 65° C. for 5 min. RNA purification and cDNA synthesis were performedas previously described (Tavano et al. 2005), using SuperScript IIIreverse transcriptase (Thermo Fisher Scientific, Waltham, Mass.) toconstruct the cDNA library.

Real-time PCR was performed on a 7500 Real Time PCR System (AppliedBiosystems, Forest City, Calif.) using SYBR Green JumpStart Taq ReadyMix(Sigma-Aldrich, St. Louis, Mo.). Primers used to detect transcripts arecontained in Table 3. Transcript levels were normalized to those ofSaro_0141 (rpoZ, coding for the RNA polymerase omega subunit).

TABLE 3 Primers used for RT qPCR of Novosphingobium aromaticivoransgenes. Transcript assayed for Primers Saro_01415′-GAGATCGCGGAAGAAACCGTGC-3′ (SEQ ID NO: 48) (rpoZ)5′-GATTTCATCCACCTCGTCGTCGTC-3′ (SEQ ID NO: 49) Saro_0205 (ligD)5′-CAACATCAAGTCGAACATCGCGGAAG-3′ (SEQ ID NO: 50)5′-CTGGTGGATCGAATGCAGCGAG-3′ (SEQ ID NO: 51) Saro_0793 (ligO)5′-GATCGAGGAATCTTCCTACGACGACTG-3′ (SEQ ID NO: 52)5′-GTTTACCACGCCGTGGAGGTTCAC-3′ (SEQ ID NO: 53) Saro_0794 (ligN)5′-CATATCGTCTGCACCGCTTCGATGTC-3′ (SEQ ID NO: 54)5′-GCAGAATGCCGAGCAGATCACG-3′ (SEQ ID NO: 55) Saro_1875 (ligL)5′-CCATGTCGTCAACACCGCATCG-3′ (SEQ ID NO: 56)5′-CATGTTCTCGGTCAGGTTCAGCAC-3′ (SEQ ID NO: 57) Saro_2091 (ligF)5′-GCTGCTGACGGTGTTCGAGAAG-3′ (SEQ ID NO: 58)5′-CTTGAACCAGTCGGTGTGATGCTC-3′ (SEQ ID NO: 59) Saro_2405 (ligP)5′-CATCGTCGAATACCTCGATGCCAAGTATC-3′ (SEQ ID NO: 60)5′-GTCCTGGCAGAAGCAGAACATCCAC-3′ (SEQ ID NO: 61) Saro_25955′-CCACGATCATGCTGGAAGAACTGCTC-3′ (SEQ ID NO: 62)5′-GATTCGAAGACGCGGAACGGTTCAG-3′ (SEQ ID NO: 63)

Determination of Chemical Oxygen Demand (COD).

Initial culture COD values were obtained either from uninoculatedmedium, or from inoculated medium that was immediately passed through a0.22 μm filter. Final COD values were obtained when cultures reachedtheir maximum cell densities. For these samples, we analyzed the COD ofboth the entire culture (cells and medium) and the filtered medium. Thedifference in COD between the unfiltered and filtered final samples isdefined as the COD of cellular biomass. Samples were diluted as neededand combined with High Range COD Digestion Solution (Hach, Loveland,Colo.). The mixtures were heated to 150° C. for 120 min to oxidize thematerials before absorbances were measured at 600 nm. Standards withknown COD values were analyzed in parallel.

Chemicals.

Vanillate, guaiacol, reduced L-glutathione (GSH), and2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) were purchased fromSigma-Aldrich (St. Louis, Mo.). erythro-Guaiacylglycerol-β-guaiacylether (erythro-GGE) was purchased from TCI America (Portland, Oreg.).

A racemic mixture of β-(2-methoxyphenoxy)-γ-hydroxypropiovanillone(MPHPV) was synthesized by dissolving erythro-GGE into ethyl acetate(Fisher Scientific), then slowly adding 1.25 molar equivalents of2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) and stirring for 30 min.The reaction was washed three times with saturated NaHCO₃ to removeDDQH₂ formed during the reaction. The MPHPV was purified via flashchromatography using hexane/ethyl acetate=1/3, as previously described(Gall and Kim et al. 2014), then crystallized from the eluent viasolvent evaporation.

Hydroxypropiovanillone (HPV) was synthesized as previously described forsynthesis of β-deoxy-α-veratrylglycerone, except using4-O-benzyl-acetovanillone as starting material, rather thanacetoveratrone (Gall and Kim et al. 2014). Synthesis of HPV required anadditional debenzylation step that was unnecessary in the synthesis ofβ-deoxy-α-veratrylglycerone.

Structural Analysis of NaGST_(Nu) and Molecular Modeling.

NaGST_(Nu) was screened for crystal formation against several commercialscreens at 277 and 293K using a TTP Labtech Mosquito® crystallizationrobot. The best diffracting ammonium acetate precipitated crystal wasobtained at 293K, by mixing 0.2 μL of protein solution (277 μM proteinpreincubated for 50 min with 10 mM GSH (neutralized with NaOH)) with 0.2μL of reservoir solution (4 M ammonium acetate buffered with 100 mMsodium acetate, pH 4.6). This crystal was mounted directly from thegrowth solution by drawing it through a layer of fomblin oil, thinningthe surrounding liquid with a paper wick, and was plunged into liquidnitrogen. The best diffracting ammonium sulfate precipitated crystal wasobtained at 293K using 0.13 μL of protein solution and 85 nL ofreservoir solution (1.35 M ammonium sulfate, 0.1 M lithium sulfate, and0.1 M bis-trispropane, pH 7.5). This crystal was cryopreserved by adding0.5 μL of a solution composed of 2 parts reservoir solution and one partneat glycerol to the droplet containing the crystal, and equilibratingfor 11 min prior to looping and plunging into liquid nitrogen.

Diffraction data were obtained at the GM/CA beam-line at ArgonneNational Laboratory with an Eiger 16M detector (Casanas et al. 2016).Data were collected on the ammonium acetate and ammonium sulfate crystalforms using 1.033 Å (for 1.45 Å resolution) or 0.7749 Å (for 1.25 Åresolution) X-rays, respectively. Diffraction data were reduced usingXDS (Kabsch 2010). Both crystals belonged to space group P2₁2₁2₁ with apredicted solvent content of 60%. The structure was solved by molecularreplacement with Phaser (McCoy et al. 2007) in the Phenix suite (Adamset al. 2010), using a search model based on PDB ID 3C8E:A (ecYghU(Stourman et al. 2011)) modified with phenix.sculptor (Bunkóczi et al.2011), based on primary sequence alignment. Structure solution revealedtwo copies of the protein per asymmetric unit, with strong electrondensity present for the paired active site glutathione molecules.Phenix.refine (Afonine et al. 2012) and COOT (Emsley et al. 2004) werealternatively used to refine the structure and fit the model to electrondensity maps.

For modeling β(R)- and β(S)-GS-HPV and their syringyl phenylpropanoidanalogues into the active sites of NaGST_(Nu) and ecYghU, the PyMOLBuilder function (The PyMOL Molecular Graphics System, Version 1.8.2.1)was used to create molecules of GS-HPV or GS-syringyl by adding atomsonto the GSH2 molecule bound in each active site. Atoms were added so asto visually minimize steric clash with the proteins. The potentialenergies of the protein-GS-phenylpropanoid complexes were minimizedusing the Minimize Structure function of UCSF Chimera (Pettersen et al.2004). For the energy minimization, all of the atoms of theprotein-GS-phenylpropanoid complex were held rigid except for those ofthe phenylpropanoid moiety and the Cys sidechain of GSH2. One-hundredsteepest descent steps were run, followed by twenty conjugate gradientsteps, and all step sizes were 0.05 Å.

Recipes for Media Components:

-   -   Hutner's vitamin-free Concentrated Base (Cohen-Bazire et        al. 1957) (per 500 mL):        -   5 g Nitrilotriacetic acid        -   14.78 g MgSO₄.7H₂O        -   1.67 g CaCl₂.2H₂O        -   4.625 mg (NH₄)₆Mo₇O₂₄.4H₂O        -   49.5 mg FeSO₄.7H₂O        -   25 mL Metals “44”    -   Metals “44” (per 500 mL):        -   1.25 g EDTA (free acid) [Add this first; use 10 M NaOH to            help dissolve it.]        -   5.475 g ZnSO₄.7H₂O        -   2.5 g FeSO₄.7H₂O        -   0.77 g MnSO₄.H₂O        -   0.196 g CuSO₄.5H₂O        -   0.125 g Co(NO₃)₂.6H₂O        -   0.0885 g Na₂B₄O₇.10H₂O

Construction of Defined N. aromaticivorans Mutants.

N. aromaticivorans has been reported to use sucrose as sole carbonsource (Stolz et al. 2000), but we found it to be incapable of growingin the presence of ≥10% sucrose (Table 2). We also noticed that the geneSaro_1879 is annotated as sacB, whose product, levansucrase, makessucrose inhibitory to growth of many Gram-negative bacteria (Gay et al.1985). To create an N. aromaticivorans strain that we could modify usinga sacB-containing plasmid, we deleted Saro_1879 from its genome. To dothis, we constructed plasmid pK18msB/ΔSaro1879, in which genomic DNAfrom upstream and downstream of Saro_1879 was cloned into pK18msB-MCS1(for details of plasmid construction, see below). This plasmid wasmobilized into N. aromaticivorans via electroporation using a single 2.5kV pulse in a 0.2 cm cuvette in a MicroPulser apparatus (Bio-Rad,Hercules, Calif.). N. aromaticivorans was made electrocompetent bywashing exponential phase cells from LB cultures twice with ice-cold 0.5M glucose, then resuspending the cells into 10% glycerol. Transformantsin which the plasmid was integrated into the genome via homologousrecombination (single crossovers) were selected for by growth on solidLB containing kanamycin. These strains were grown in liquid LB to allowplasmid loss via homologous recombination, then plated on solid LBcontaining 10% sucrose to select for sucrose-tolerance. Sucrose-tolerantstrains in which Saro_1879 had been deleted from the genome wereconfirmed by PCR amplification and sequencing of genomic DNA. One ofthese strains (12444Δ1879) was used as the parent strain for subsequentgenetic modifications.

To inactivate Saro_2595, we electroporated plasmid pK18msB/ΔSaro2595 (inwhich genomic DNA from upstream and downstream of Saro_2595 was clonedinto pK18msB-MCS1) into strain 12444Δ1879, and a strain lacking theSaro_2595 gene (referred to as 12444Δ2595) was isolated via the processdescribed above for deleting Saro_1879.

To generate a strain (referred to as 12444ecyghU) in which Saro_2595 wasreplaced in the N. aromaticivorans genome with the E. coli yghU gene, weconstructed plasmid pK18msB/ecyghU-42595, in which yghU (amplified fromE. coli DH5α genomic DNA) was cloned into pK18msB/ΔSaro2595. Thisplasmid was mobilized into 12444Δ2595 via conjugation from E. coliS17-1. Transconjugants (single crossovers) of N. aromaticivorans wereisolated on solid GluSis containing kanamycin. After growth in liquidGluSis, strains that lost the plasmid and retained the yghU gene at thenative Saro_2595 locus were isolated on solid GluSis medium containingsucrose. The presence of the yghU gene in the genome was confirmed viaPCR and sequencing.

Purification of Recombinant Proteins.

Recombinant proteins were purified as described previously (Gall and Kimet al. 2014), except that cells were lysed by sonication, first using aBranson Sonifier 450 (Branson Ultrasonics, Danbury, Conn.) (duty cycle50%, output level 6, for six rounds of 1-2 min), then a Qsonica Q500with a cup horn attachment (Qsonica, Newtown, Conn.) (60% amplitude for10 minutes with cycles of 10 s on, 10 s off). His-tagged proteins werepurified from lysates over a column packed with Ni²⁺-NTA resin (Qiagen,Hilden, Germany) attached to an AKTAprime plus FPLC (GE Healthcare LifeSciences), then incubated with TEV protease to remove the His-tag. Theprotease reaction mixture was passed through a Ni²⁺-NTA column toseparate the recombinant protein from the cleaved His₈-tag and TEVprotease (which also contained a His-tag).

Procedures to Construct Plasmids for Genomic Modifications for N.aromaticivorans and for Purifying Enzymes

Biological reagents. All PCR reactions were performed with Herculase IIpolymerase (Agilent Technologies, Santa Clara, Calif.). Primers werephosphorylated with polynucleotide kinase from Promega (Madison, Wis.).All other enzymes were from New England Biolabs (Ipswich, Mass.). Allprimers were from Integrated DNA Technologies (Coralville, Iowa). SeeTable 4.

TABLE 4 Primers used in genomic modifications and enzyme expression.Name Sequence Notes pK18msB AseI 5′-CTGTCGTGCCAGCTGCATTAATG-3′ (SEQ AseIsite ampl F ID NO: 64) (underlined) native to template pK18msB-5′-GAACAtcTAGAAAGCCAGTCCGCAGAA XbaI site MCS XbaI R AC-3′ (SEQ ID NO:65) (underlined); lowercase bases do not match template Saro18795′-CCCGAattaATCGTGACGGTATCAACCT AseI site lvnsucr ampl F CC-3′ (SEQ IDNO: 66) (underlined); AseI lowercase bases do not match templateSaro1879 5′-GTTTCGGtCtAGATCGAGCTGACCGAA XbaI site lvnsucr ampl RATC-3′ (SEQ ID NO: 67) (underlined); XbaI lowercase bases do not matchtemplate Saro_2595 5′-GTCGatTAatAGTCCGAGATCGAGGC AseI site amp AseI forTGC-3′ (SEQ ID NO: 68) (underlined); lowercase bases do not matchtemplate Saro_2595 5′-CGACtctAGaCAGAGCCTGAACGAA XbaI site amp XbaI revGTC-3′ (SEQ ID NO: 69) (underlined); lowercase bases do not matchtemplate Saro1879 5′-CCGACTTTCTTGAAACAGATTTGGCTT lvnsucr delAAGAC-3′ (SEQ ID NO: 70) REV Saro1879 5′-GTTCATGCTTAACTTCGATGGCGAGC-3′lvnsucr del (SEQ ID NO: 71) FOR Saro_2595 del5′-CCTGCTCCTTGGGGATATTGTTAGTG rev TTG-3′ (SEQ ID NO: 72) Saro_2595 del5′-GGAATCGTTGCAAGCGATCGTCAAG-3′ for (SEQ ID NO: 73) D2595pK18-5′-GGAGCAGGCGATGACAGACAATACTT underlined region ecYghU FATCAGCCCGCGAAAG-3′ (SEQ ID NO: 74) matches sequence in pK18msB/ΔSaro2595D2595pK18- 5′-CGAGGCGGGTTTACCCCTGACGCTTAT underlined region ecYghU RCTTCCGTATTCGTC-3′ (SEQ ID NO: 75) matches sequence in pK18msB/ΔSaro2595ecYghU- 5′-TCAGGGGTAAACCCGCCTCGAGACCG underlined region D2595pK18 FGCGAAC-3′ (SEQ ID NO: 76) matches sequence in yghU ecYghU-5′-TGTCTGTCATcgCCTGCTCCTTGGGGAT underlined region D2595pK18 RATTGTTAGTG-3′ (SEQ ID NO: 77) matches sequence in yghU; lowercase basesare not present in pK18msB/ΔSaro2595, but are present in the N.aromaticivorans genome Saro2595 Ctag 5′-GCAGGacATGTCCTCAGAGTACGTTCC-3′PciI site PciI F (SEQ ID NO: 78) (underlined); lowercase bases do notmatch template Saro2595 Ctag 5′-GTTatctgcgagaccACGATCGCTTGCAACG BsaIsite BsaI R ATTC-3′ (SEQ ID NO: 79) (underlined); lowercase bases do notmatch template pVP302K Ctag 5′-CTGCGGTCTCGCAGATGGTAAAATT BsaI site BsaIF CTG-3′ (SEQ ID NO: 80) (underlined) pVP302K Ctag5′-GGTGATGTCCCATGGTTAATTTCTCCTC NcoI site NcoI R TTTAATG-3′ (SEQ ID NO:81) (underlined) Ctag 2595-pVP 5′-tcagaagcccttgACGATCGCTTGCAACGAlowercase bases do add Stop R TTC-3′ (SEQ ID NO: 82) not match pCtag-2595/pVP302K; underlined bases are stop codon pVP302K Ntag5′-CATTAAaAGcTTAAACGAATTCGGACT HindIII site HindIII F CGGTACGC-3′ (SEQID NO: 83) (underlined); lowercase bases do not match template 2595-pVPC to 5′-caagcgaaaatctgtattttcagagcgcgatcgcaggaATG lowercase bases doNtag F TCCTCAGAGTACGTTCC-3′ (SEQ ID NO: 84) not match template; bold ATGis Saro_2595 start site; underlined region is Tev protease recognitionsite pVP302 C to 5′-ccaatgcatggtgatggtgatgatggtgatgtcccatGGTT lowercasebases do Ntag R AATTTCTCCTCTTTAATG-3′ (SEQ ID NO: 85) not matchtemplate; compliment of coding region for 8X-Histidine tag is underlinedpVP302K- 5′-GATCGCAGGAATGACAGACAATACTT underlined region ecYghU FATCAGCCCGCGAAAG-3′ (SEQ ID NO: 86) matches sequence in pVP302K pVP302K-5′-CGGCTTTCTGTTACCCCTGACGCTTATC underlined region ecYghU RTTCCGTATTCGTC-3′ (SEQ ID NO: 87) matches sequence in pVP302K ecYghU-5′-TCAGGGGTAACAGAAAGCCGAAAATA underlined region pVP302K FACAAAGTTAGCCTGAGCTG-3′ (SEQ ID matches sequence in NO: 88) yghU ecYghU-5′-TGTCTGTCATTCCTGCGATCGCGCTCT underlined region pVP302K RGAAAATACAGATTTTCG-3′ (SEQ ID NO: 89) matches sequence in yghU pVP302K-5′-TCCTGCGATCGCGCTCTGAAAATACAG HiFi-ATW-R ATTTTCG-3′ (SEQ ID NO: 90)pVP302K- 5′-CAGAAAGCCGAAAATAACAAAGTTAG HiFi-ATW-F CCTGAGCTG-3′ (SEQ IDNO: 91) ecYfcG-pVP- 5′- lowercase region Ntag-HiFi-FgtattttcagagcgcgatcgcaggaATGATCGATCTCTA complementary toTTTCGCCCCGACAC-3′ (SEQ ID NO: 92) “pVP302K-HiFi- ATW-R” ecYfcG-pVP- 5′-lowercase region Ntag-HiFi-R ctaactttgttattttcggctttctgTTAACTATCCGAACGCcomplementary to TCATCACCGAGTTG-3′ (SEQ ID NO: 93) “pVP302K-HiFi- ATW-F”SYK6 yghU 5′-gttattttcggctttctgttaagCTTCGGTCTTCG-3′ Lowercase region pVPfix R (SEQ ID NO: 94) complementary to “SYK6 yghU pVP fix F” SYK6 yghU5′-cttaacagaaagccgaaaataacAAAGTTAGCCT Lowercase region pVP fix FGAG-3′ (SEQ ID NO: 95) complementary to “SYK6 yghU pVP fix R”

Construction of pK18msB-MCS1.

Plasmid pK18mobsacB (Schafer et al. 1994) was amplified via PCR withphosphorylated primers “pK18msB AseI ampl F” and “pK18msB-MCS XbaI R”.The product was circularized with T4 DNA ligase, then transformed intoE. coli DH5α. The final 5278-base pair (bp) plasmid, pK18msB-MCS1, issimilar to pK18mobsacB, except that the multiple cloning site has beenremoved, and a new XbaI site is 24 bp from one of the plasmid's nativeAseI sites (with the other native AseI site removed).

Plasmids for Deleting Saro_1879 or Saro_2595.

Regions of N. aromaticivorans genomic DNA containing either Saro_1879 orSaro_2595, along with ˜1300-1600 bp flanking regions upstream anddownstream of each gene, were separately amplified via PCR using theprimer pairs “Saro1879 lvnsucr ampl F AseI”/“Saro1879 lvnsucr ampl RXbaI” and “Saro_2595 amp AseI for”/“Saro_2595 amp XbaI rev”. Theamplified DNA fragments were digested with AseI and XbaI, then ligatedwith T4 DNA ligase into AseI- and XbaI-digested pK18msB-MCS1. Theresulting plasmids (pK18msB/Saro1879 and pK18msB/Saro2595) weretransformed into E. coli (strain DH5α for the Saro_1879 plasmid andTurbo for the Saro_2595 plasmid). PCR was performed on the purifiedplasmids using the phosphorylated primer pairs “Saro1879 lvnsucr delREV”/“Saro1879 lvnsucr del FOR” or “Saro_2595 del rev”/“Saro_2595 delfor” to generate linear plasmids lacking the majority of the Saro_1879and Saro_2595 coding regions, respectively. These DNA fragments werecircularized with T4 DNA ligase to form plasmids pK18msB/ΔSaro1879 andpK18msB/ΔSaro2595.

Plasmid for Incorporating E. coli yghU into the N. aromaticivoransGenome.

The yghU gene (locus tag Ga0077588_1407) was amplified from E. coli DH5αgenomic DNA using primers “D2595pK18-ecYghU F” and “D2595pK18-ecYghU R”,which each contain a sequence on their 5′ end that is complementary to aregion in the plasmid pK18msB/ΔSaro2595. pK18msB/ΔSaro2595 was amplifiedwith the primers “ecYghU-D2595pK18 F” and “ecYghU-D2595pK18 R”, whicheach contain a sequence on their 5′ ends that is complementary to E.coli yghU, to generate a linear fragment in which pK18msB-MCS1 containsthe regions flanking Saro_2595 in the N. aromaticivorans genome, alongwith the regions complementary to yghU.

The PCR amplified fragments were connected using NEBuilder HiFi DNAAssembly Master Mix (New England Biolabs) (using 81 ng of the linearpK18msB/ΔSaro2595 fragment and 22 ng of the yghU fragment) andtransformed into NEB 5-alpha competent E. coli (New England Biolabs).The resulting plasmid (pK18msB/ecyghU-Δ2595) consisted of pK18msB-MCS1containing the E. coli yghU gene flanked by regions that flank Saro_2595in the N. aromaticivorans genome (i.e., with the start and stop codonspositioned where the respective codons of Saro_2595 would naturally be).

Enzyme Expression Plasmids

Recombinant Saro_2595.

Saro_2595 was amplified from N. aromaticivorans genomic DNA with theprimers “Saro2595 Ctag PciI F” and “Saro2595 Ctag BsaI R”. This fragmentwas digested with PciI and BsaI. The expression vector pVP302K (Gall andRalph et al. 2014) was amplified using the primers “pVP302K Ctag BsaI F”and “pVP302K Ctag NcoI R”. This fragment was digested with BsaI andNcoI. The digested fragments were ligated using T4 DNA ligase,generating plasmid pVP302K/Ctag-2595, which consists of a T5 promoterfollowed by the coding sequences of Saro_2595, the RtxA protease fromVibrio cholerae, and an 8X-Histidine tag.

pVP302K/Ctag-2595 was amplified using phosphorylated primers “Ctag2595-pVP add Stop R” and “pVP302K Ntag HindIII F”. This fragment wascircularized using T4 DNA ligase to generate plasmidpVP302K/Untagged2595, in which a stop codon has been introduced directlyafter Saro_2595. pVP302K/Untagged2595 was amplified via PCR usingphosphorylated primers “2595-pVP C to Ntag F” and “pVP302 C to Ntag R”.This fragment was circularized using T4 DNA ligase to generate plasmidpVP302K/Ntag-2595, which contains a T5 promoter followed by codingsequences for a His₈ tag, a tobacco etch virus (Tev) proteaserecognition site and Saro_2595.

Recombinant E. coli YghU.

The yghU gene was amplified from E. coli DH5a genomic DNA using theprimers “pVP302K-ecYghU F” and “pVP302K-ecYghU R”. pVP302K was amplifiedby PCR using the primers “ecYghU-pVP302K F” and “ecYghU-pVP302K R”. Thetwo amplified fragments, with ends that are complementary to each other,were concurrently transformed (94 ng linear pVP302K, 168 ng yghU gene,in 4 μL TE buffer) into E. cloni 10G chemically competent cells(Lucigen, Middleton, Wis.). The fragments were combined via homologousrecombination in vivo (Bubeck et al. 1993), and the resulting plasmid,pVP302K/Ntag-ecyghU, was purified from the cells and verified via Sangersequencing.

Recombinant E. coli YfcG.

The yfcG gene was amplified from E. coli DH5a genomic DNA using theprimers “ecYfcG-pVP-Ntag-HiFi-F” and “ecYfcG-pVP-Ntag-HiFi-R”. pVP302Kwas amplified via PCR using the primers “pVP302K-HiFi-ATW-R” and“pVP302K-HiFi-ATW-F”. The two amplified fragments, with ends that arecomplementary to each other, were combined using NEBuilder HiFi DNAAssembly Master Mix (New England Biolabs) (100 ng linear pVP302K, 48 ngyghU gene) and transformed into NEB 5-alpha competent E. coli (NewEngland Biolabs). The resulting plasmid, pVP302K/Ntag-ecyfcG, waspurified and verified via sequencing.

Recombinant SLG_04120.

A fragment containing the SLG_04120 gene, codon-optimized for E. coli,with ends complementary to pVP302K, was ordered as a gBlock from NewEngland Biolabs. The sequence of the fragment was:

(SEQ ID NO: 96) 5′-GTATTTTCAGAGCGCGATCGCAGGAATGGCCGACTCAGATCCATCCATGAATCAGCCGACGGGTTACGTCCCGCCGAAAGTTTGGACCTGGGACAAAGAGAACGGCGGTCAGTTCAGCAATATCAACGCCCCTACGGCTGGTGCGCGCCAGGACGTCACGCTCCCTGTAGGGGAGCACCCTATCCAATTATATAGTCTCGGCACTCCGAATGGTCAGAAAGTTACTATCATGTTGGAAGAACTGCTGGCTGCTGGCTTTGATGCTGAGTATGACGCCTGGCTCATCAAAATCTACACAGGCGAGCAATTCGGATCTGATTTCGTCGCCATTAACCCTAATAGCAAAATTCCGGCTATGATGGACCATGGTCTCGATCCGCCGCTCCGTTTATTTGAGTCTGGTTCTATGTTAGTTTATCTGGCCGAAAAGTTTGGCGCATTCCTCCCGACCGAAATCCGCAAACGTACGGAAACCTTTAACTGGCTCATGTGGCAGATGGGTTCTGCTCCTTTTGTGGGTGGTGGCTTTGGCCACTTCTATGCGTACGCCCCATTTAAAATCGAATATGCCATTGATCGTTACGCGATGGAAACCAAGCGCCAACTGGACGTTCTGGATAAAAATCTGGCCGATCGTGAATTTATGATCGGCGATGAAATCACCATCGCAGATTTTGCGATTTTCCCTTGGTACGGCTCGATTATGCGTGGCGGTTACAACGCGCAAGAATTCTTGAGCACTCACGAGTACCGTAACGTTGATCGCTGGGTTACGCAGCTTTCTGAACGTACGGGCGTAAAGCGTGGTCTCCTTGTCAATTCCGCGGGTCGCCCGGGAGGTGGCATTGCGGAACGCCATAGCGCGGCTGATTTAGACGCGTCGATTAAAGCGGCTGAACAAGAGGCCGCGAAGACCGAAGCTtAACAGAAAGCCGAAAATAACAAAG TTAG-3′ (underlinedregions match sequences in pVP302K)

pVP302K was amplified via PCR using the primers “pVP302K-HiFi-ATW-R” and“pVP302K-HiFi-ATW-F”.

The amplified pVP302K fragment was combined with SLG_04120 gBlock usingligation-free cloning: fragments were mixed (57 ng linear pVP302K, 84 ngSLG_04120, in 4 μL TE buffer), then transformed into E. cloni 10Gcompetent cells (Lucigen, Middleton, Wis.). DNA sequencing was used toidentify a plasmid containing the correct DNA sequence for SLG_04120 inpVP302K, resulting in plasmid pVP302K/Ntag-SLG_04120.

Identification and Quantification of Extracellular Metabolites

Initial Identification Using LC-MS.

Compounds were separated on a Phenomenex PFP 250×4.6-mm column attachedto an Accela LC pump equipped with a PDA UV detector. Running buffers A(5 mM formic acid and 5% acetonitrile in H₂O) and B (methanol) wereinitially at 82.5% and 17.5%, respectively. Buffer B was held at 17.5%for 18 minutes, then increased to 50% over 5 minutes, held at 50% for 3minutes, then returned to initial conditions and re-equilibrated for 4minutes. Flow rate was 1 mL per minute. UV absorbance data from 190-500nm at 5 Hz and single wavelength data at 254 nm (20 Hz, 9 nm bandwidth)were collected.

Samples were analyzed by high resolution, tandem mass spectrometry usinga Thermo Scientific Q Exactive Orbitrap mass spectrometer. The massspectrometer was operated in fast polarity switching mode withacquisition of MS/MS spectra of the two most abundant precursor ionsfrom the preceding MS1 scan (50-750 Th). Resolution was 35,000 at 200 Thfor MS1 scans and 17,500 at 200 Th for MS/MS scans. Capillary voltagewas set at 4000V in both polarities, sheath gas at 50 units, auxiliarygas at 20 units, probe heater at 350° C., inlet capillary at 325° C.,and the S-Lens at 50 units. AGC target was 1e6 for MS1 scans and 2e5 forMS/MS scans with a maximum injection time of 50 ms. The isolation widthfor MS/MS scans was set to 2 Th and a 5 s dynamic exclusion time wasused.

Elemental compositions of the metabolites were derived from the massmeasurements. From the MS/MS fragmentation patterns and previous data,we provisionally identified the metabolites. We used standards toconfirm these putative identifications by matching retention times andmass spectra.

Results

GGE Metabolism by Sphingomonads.

Genomic sequences predict (Ohta et al. 2015) that N. aromaticivorans DSM12444 (Fredrickson et al. 1991, Fredrickson et al. 1995),Novosphingobium sp. PP1Y (Notomista et al. 2011), and Sphingobiumxenophagum NBRC 107872 (Stolz et al. 2000) contain genes that encodeenzymes required to metabolize guaiacylglycerol-β-guaiacyl (GGE) via thebacterial β-etherase pathway (FIG. 1). To test this, we fed thesebacteria erythro-GGE (FIG. 1), either as sole carbon source or in thepresence of another organic molecule.

We found that N. aromaticivorans 12444Δ1879 metabolized erythro-GGE andassimilated it into cell material when fed GGE alone (FIG. 2A (panelsA,B) and FIG. 3) or GGE plus vanillate (FIG. 2B (panels G,H) and Table5). β-Etherase pathway intermediates threo-GGE, MPHPV, and HPVtransiently appeared in the media of both cultures (FIGS. 2A and 2B(panels B,H) and FIG. 3), whereas the pathway intermediate guaiacol wasonly detected in the medium of the culture fed GGE plus vanillate (FIG.2B (panel H)). The predicted pathway intermediate GS-HPV was notdetected in the medium of either culture.

Compared to N. aromaticivorans, Novosphingobium sp. PP1Y grewsignificantly slower in medium containing only GGE (FIG. 4A (panelsA,B)). The maximum cell density and amount of COD incorporated intobiomass were the same when Novosphingobium sp. PP1Y was fed either GGEplus vanillate or vanillate only (FIG. 4A (panels E,F) and Table 5),suggesting that this strain did not convert a significant amount of GGEinto biomass in the presence of vanillate. S. xenophagum did notassimilate GGE into cell material in any culture tested (based on celldensity and COD measurements; FIG. 4B (panels C,G) and Table 5),although low levels of some β-etherase pathway intermediates wereobserved in its culture media (FIG. 4B (panels D,H)).

TABLE 5 Chemical oxygen demand (COD) analysis of bacterial cultures.^(a)% COD % COD lost from Initial Final COD Final COD incorporated theStrain Carbon souces COD^(b) (biomass)^(c) (soluble)^(d) intobiomass^(e) cultue^(f) N. aromaticivorans 3 mM GGE 2100 ± 100 480 ± 70500 ± 100 22% 53% 12444Δ1879 (FIG. 2A (A, B)) 4 mM vanillate (FIG. 1300± 100 530 ± 40 280 ± 80  41% 37% 2B (G)) 4 mM vanillate, 2170 ± 80  720± 80 340 ± 60  33% 51% 15 mM GGE (FIG. 2B (G, H)) Novophingobium sp. 4mM vanillate (FIG. 1200 ± 100 420 ± 20 240 ± 30  36% 44% PP1Y 4A (E)) 4mM vanillate, 2070 ± 90  420 ± 20 1080 ± 20  20% 28% 15 mM GGE (FIG. 4A(E, F) S. xenophagum NBRC 4 mM glucose 1040 ± 30  490 ± 30 340 ± 20  47%20% 107872 (FIG. 4B (G)) 4 mM glucose, 1970 ± 50  460 ± 30 1270 ± 30 23% 12% 15 mM GGE (FIG. 4B (G, H)) N. aromaticivorans 3 mM GGE 2090 ±40   30 ± 100 1900 ± 30   1%  6% 12444Δ2595 (FIG. 2A (C, D)) 4 mMvanillate (FIG. 1300 ± 200  550 ± 100 330 ± 70  41% 34% 2B (I)) 4 mMvanillate, 2200 ± 100 520 ± 80 1200 ± 100  23% 24% 15 mM GGE (FIG. 2B(I, J)) N. aromaticivorans 3 mM GGE 2200 ± 90   500 ± 100 500 ± 100 23%52% 12444ecyghU (FIG. 2A (E) 4 mM vanillate (FIG. 1400 ± 300 570 ± 50430 ± 50  42% 26% 2B (K)) 4 mM vanillate, 2400 ± 100  600 ± 100 450 ±80  26% 55% 15 mM GGE (FIG 2B (K, L)) ^(a)Units of COD are mg/L^(b)Initial COD is that of the medium before inoculation. ^(c)Final COD(biomass) is the difference between the unfiltered and filtered finalsamples. ^(d)Final COD (soluble) is the COD remaining in the mediumafter filtering the final sample. ^(e)% COD incorporated into biomass isthe ratio of Final COD (biomass) to Initial COD. ^(f)% COD lost = 1 −(Final COD (biomass) + Final COD (soluble))/Initial COD. It is assumedthat the lost COD represents the electrons in the system that werecombined with oxygen during cell growth.

Transcripts of Predicted β-Etherase Pathway Genes Increase in Abundancewhen N. aromaticivorans Grows in the Presence of GGE.

Since N. aromaticivorans metabolized GGE, we investigated the expressionof genes predicted to be involved in the β-etherase pathway in thisorganism. With the exception of ligL, transcription levels of the geneswe tested were increased in cells grown in the presence of GGE versusits absence (Table 6). Among the GGE-induced transcripts was one derivedfrom Saro_2595, which encodes a Nu-class glutathione-S-transferase(named here NaGST_(Nu)).

TABLE 6 Fold-changes of transcript levels in N. aromaticivorans culturesgrown in vanillate with or without GGE.^(a) Gene Homologue^(b)Fold-change^(c) Saro_0205 SLG_08640, LigD (77.6%) 11 ± 3  Saro_0793SLG_35880, LigO (41.5%) 6 ± 2 Saro_0794 SLG_35900, LigN (44.7%) 9 ± 1Saro_1875 SLG_33660, LigL (48.7%) 1 ± 1 Saro_2091 SLG_08650, LigF(59.1%) 8 ± 1 Saro_2405 SLG_32600, LigP (65.9%) 17 ± 9  Saro_2595MBENS4_2527, GST3 (37.6%) 8 ± 2 ^(a)Transcript levels for each culturewere normalized to those of Saro_0141 (rpoZ). ^(b)Homologue is the genethat codes for a product from Sphingobium sp. SYK-6 (SLG) orNovosphingobium sp. MBES04 (MBENS4) with the highest % amino acididentity to the product of the indicated N. aromaticivorans (Saro) gene.^(c)Fold-change is the ratio of the normalized transcript level in cellsgrown in the presence of GGE to that in cells grown in the absence ofGGE.

NaGST_(Nu) cleaves β(R)- and β(S)-GS-HPV.

NaGST_(Nu) is 38% identical in amino acid sequence to Novosphingobiumsp. MBESO4 GST3 (FIG. 5), which can cleave both β(R)- and β(S)-GS-HPVinto HPV in vitro (FIG. 1; (Ohta et al. 2015)). Since N. aromaticivoranslacks any homologues of LigG (the enzyme from Sphingobium sp. SYK-6 thatcleaves the β(R)-stereoisomer of GS-HPV (FIG. 1; (Masai et al. 2003)),we tested whether NaGST_(Nu) could cleave both β(R)- and β(S)-GS-HPV inN. aromaticivorans. We found that recombinant NaGST_(Nu) cleaved bothβ(R)- and β(S)-GS-HPV in vitro (FIG. 6). Kinetic analysis of NaGST_(Nu)showed that it had slightly higher k_(cat) and ˜5-fold higher K_(M) withβ(R)-GS-HPV than with β(S)-GS-HPV, resulting in a ˜4-fold higherk_(cat)/K_(M) with the β(S)-isomer (Table 7).

TABLE 7 Kinetic parameters for the enzymatic conversion of GS-HPV intoHPV.^(a) GS- k_(cat) K_(M) k_(cat)/K_(M) Protein HPV^(b) (s⁻¹) (μM)(mM⁻¹s⁻¹) NaGST_(Nu) (8 nM) β(R) 80 ± 10 40 ± 6  1900 ± 400 SYK6GST_(Nu)(47 nM) β(R) 13 ± 1  55 ± 7  240 ± 40 ecYghU (195 nM) β(R) 0.43 ± 0.0328 ± 4  16 ± 3 ecYfcG (195 nM) β(R) 0.04 ± 0.01 160 ± 60   0.2 ± 0.1NaGST_(Nu) (8 nM) β(S) 57 ± 9  8 ± 3  8000 ± 3000 SYK6GST_(Nu) (18 nM)β(S) 30 ± 5  11 ± 2  2700 ± 700 ecYghU (195 nM) β(S) 0.29 ± 0.03 12 ± 3 24 ± 6 ecYfcG (195 nM) β(S) 0.017 ± 0.004 130 ± 40   0.14 ± 0.06^(a)Kinetic data are from fits shown in FIG. 6 ^(b)Stereoisomer ofGS-HPV used in reaction (see FIG. 1)

NaGST_(Nu) is Necessary for GGE Metabolism In Vivo.

To test for an in vivo role of NaGST_(Nu), we generated an N.aromaticivorans mutant lacking Saro_2595 (12444Δ2595). Unlike its parentstrain (12444Δ1879), 12444Δ2595 did not incorporate significant GGE intocell material in any culture (based on cell density and CODmeasurements; FIGS. 2A and B (panels C,I)) and Table 5). When providedonly erythro-GGE, 12444Δ2595 produced only a small amount of MPHPV andthreo-GGE in the medium (FIG. 2A (panel D)). When 12444Δ2595 was fedboth vanillate and erythro-GGE, it completely metabolized the vanillate,and converted almost all of the GGE into MPHPV (FIG. 2B (panel J)). Asmall amount of guaiacol also appeared in the medium of this culture,suggesting that some MPHPV was cleaved by 12444Δ2595. However, unlikethe situation for the parent strain, no extracellular HPV was detectedin the 12444Δ2595 culture (FIGS. 2A and 2B (panels D,J)). These resultsshow that NaGST_(Nu) is necessary for complete GGE metabolism by N.aromaticivorans.

NaGST_(Nu) is Sufficient for Conversion of GS-HPV into HPV in N.aromaticivorans.

To determine which step in the β-etherase pathway requires NaGST_(Nu),we incubated cell extracts of 12444Δ2595 and its parent strain(12444Δ1879) with racemic MPHPV and GSH. With the 12444Δ1879 extract,MPHPV was converted to roughly equimolar amounts of guaiacol and HPV,along with a small amount of GS-HPV (FIG. 7 (A)). In contrast, the12444Δ2595 extract incompletely cleaved MPHPV, producing roughlyequimolar amounts of guaiacol and GS-HPV along with a low level of HPV(˜2% of the level of GS-HPV formed; FIG. 7 (B)). Thus, it appeared thatthe 12444Δ2595 extract was defective in the conversion of GS-HPV intoHPV. When recombinant NaGST_(Nu) was added to the 12444Δ2595 extract(FIG. 7 (B)), the GS-HPV disappeared, with a concomitant increase inHPV, showing that the defect in GS-HPV cleavage by 12444Δ2595 extractwas caused by the lack of NaGST_(Nu).

NaGST_(Nu) Homologues Cleave β(R)- and β(S)-GS-HPV.

The GST Nu-class is a widespread protein family (Stourman et al. 2011,Mashiyama et al. 2014). Indeed, a non-redundant BLAST search of the NCBIdatabase using NaGST_(Nu) as query identified over 1,000 proteins withamino acid identities of >61% and E-values <2.0×10⁻¹²⁴. Besides GST3from Novosphingobium sp. MBESO4 (Ohta et al. 2015), the only otherNu-class GSTs that have been analyzed for catalytic activity are E. coliecYghU and ecYfcG (61% and 42% amino acid sequence identity withNaGST_(Nu), respectively; FIG. 5). The roles of these enzymes areunknown, but they are reported to have disulfide bond oxidoreductaseactivity in vitro (Stourman et al. 2011, Wadington et al. 2009,Wadington et al. 2010, Mashiyama et al. 2014). Though E. coli is notknown to metabolize lignin-derived GSH adducts, we found thatrecombinant ecYghU and ecYfcG were both able to cleave β(R)- andβ(S)-GS-HPV in vitro (FIG. 6). While the K_(M) values for ecYghU werecomparable to those of NaGST_(Nu) (Table 7), its k_(cat) values weremuch lower than those of NaGST_(Nu), resulting in k_(cat)/K_(M) valuesfor ecYghU with GS-HPV ˜100-fold lower than those of NaGST_(Nu) underour assay conditions (Table 7). For ecYfcG, K_(M) values were ˜10-foldhigher and k_(cat) values were ˜10-fold lower than those of ecYghU,leading to k_(cat)/K_(M) values with GS-HPV ˜100-fold lower for ecYfcGthan for ecYghU.

To test whether ecYghU can function in the β-etherase pathway in vivo,we created a strain of N. aromaticivorans in which Saro_2595 wasreplaced in the genome by the E. coli yghU gene. The resulting strain,12444ecyghU, metabolized GGE slower than 12444Δ1879 (the straincontaining NaGST_(Nu); FIGS. 2A and 2B), but still removed all of theGGE from the medium and assimilated it into biomass, whereas 12444Δ2595could not (FIGS. 2A and 2B (panels E,F,K,L); Table 5). This shows thatecYghU can substitute for NaGST_(Nu) in the N. aromaticivoransβ-etherase pathway.

Although the sphingomonad Sphingobium sp. SYK-6 can metabolize GGE(Palamuru et al. 2015, Sato et al. 2009), no enzyme capable of cleavingβ(S)-GS-HPV has been identified in this organism (Masai et al. 2003). Wetested a recombinant version of the Nu-class GST from Sphingobium sp.SYK-6, coded for by SLG_04120 and named here SYK6GST_(Nu), and foundthat it cleaved both β(S)- and β(R)-GS-HPV in vitro (FIG. 6).SYK6GST_(Nu) had higher k_(cat) and lower K_(M) with β(S)-GS-HPV thanwith β(R)-GS-HPV, leading to a ˜10-fold greater k_(cat)/K_(M) with theβ(S)-isomer (Table 7). Thus, SYK6GST_(Nu) could cleave β(S)-GS-HPV andpotentially contribute, along with LigG, to β(R)-GS-HPV cleavage inSphingobium sp. SYK-6 (Masai et al. 2003).

Structural Characterization of NaGST_(Nu).

We solved two structures of NaGST_(Nu), crystallized under differentconditions, with resolutions of 1.25 (pdb 5uuo) and 1.45 (pdb 5uun) Å(Table 8). The structures align with each other with an RMS distance of0.108 over 7381 atoms. NaGST_(Nu) is a homo-dimer; each subunit containsa characteristic N-terminal GST (thioredoxin-like) domain (Val39 toGly129), a C-terminal GST domain (Ser135 to Leu257), and a C-terminalextension not present in most other characterized classes of GSTs(Val258 to Phe288)* (FIG. 8 (A)).

TABLE 8 Statistics for the crystal structure determinations ofNaGST_(Nu). PDB entry 5uuo 5uun Precipitant Ammonium sulfate Ammoniumacetate Wavelength 0.7749 1.033 Resolution range 29.36-1.25 (1.295-1.25)43.97-1.45 (1.502-1.45) Space group P 21 21 21 P 21 21 21 Unit cell68.81 70.39 68.59 70.64 168.23 90 90 90 168.57 90 90 90 Totalreflections 3049254 (309691) 1854065 (113574) Unique reflections 225346(22262) 140421 (11067) Multiplicity 13.5 (13.9) 13.2 (10.3) Completeness(%) 99.91 (99.70) 96.48 (76.88) Mean I/sigma(I) 23.67 (1.96) 36.36(8.82) Wilson B-factor 16.03 13.23 R-Merge 0.05235 (1.297) 0.04367(0.2047) R-Means 0.05445 (1.346) 0.04544 (0.2159) R-Pim 0.01485 (0.3565)0.01239 (0.06599) CC1/2 1 (0.841) 1 (0.986) CC* 1 (0.956) 1 (0.997)Reflections used 225262 (22242) 140408 (11066) in refinement Reflectionsused 1963 (197) 1852 (154) for R-free R-Work 0.1303 (0.2607) 0.1357(0.206) R-Free 0.1324 (0.2979) 0.1365 (0.1595) CC(work) 0.978 (0.913)0.978 (0.971) CC(free) 0.988 (0.851) 0.975 (0.9363) Non-H atoms 56585852 Macromolecules 4626 4628 Ligands 252 244 Solvent 780 980 Proteinresidues 569 1040 RMS(bonds) 0.007 0.008 RMS(angles) 1.02 1.08Ramachandran 93.75/2.30/0.35 96.82/2.65/0.53 favored/allowed/ outliers(%) Rotamer 0.43 0.43 outliers (%) Clashscore 1.89 2.13 Average B-factor22.39 19.61 B-Factor 20.02/26.98/34.97 16.91/19.017/33.64macromolecules/ ligands/solvent No. TLS groups 9 9 *The residue numbersused here for NaGST_(Nu) are for the native protein represented by SEQID NO: 18; residues numbers in the pdb entries differ by +5 since theprotein used for crystallization contained an N-terminal extension leftafter proteolytic removal of the His₈-tag. Statistics for thehighest-resolution shell are shown in parentheses.

The structure of NaGST_(Nu) is most similar to those of the Nu-classGSTs ecYghU (pdb 3c8e (Stourman et al. 2011), with an RMSD of 0.49 Åover 3116 atoms) and Streptococcus sanguinis SK36 YghU (ssYghU; pdb 4mzw(Patskovsky et al.), with an RMSD of 0.58 Å over 3004 atoms). InNaGST_(Nu), ecYghU, and ssYghU, a short channel leads from the activesite pocket to the solvent (FIGS. 9 and 12). In other structurallycharacterized Nu-class GSTs, including ecYfcG (pdb 3gx0 (Wadington etal. 2009)), the active site is more solvent exposed, since theseproteins lack N-terminal residues that contribute to the channel wallsin the other proteins (FIG. 5; Supplemental Materials of Thuillier etal. 2013).

The active site electron density in NaGST_(Nu) was modeled as a mixedpopulation of GSH1 and GSH2 thiols with an S—S distance of 2.4 Å (˜60%occupancy) and GS-SG disulfide with an S—S distance of 2.0 Å (˜40%occupancy) (FIG. 8 (B)). Since the crystallization solutions initiallycontained only GSH, the disulfide likely formed via adventitiousoxidation of the closely situated GSH thiols during the crystallizationperiod (˜4 weeks), or during X-ray diffraction data collection. Forcomparison, ecYghU shows a dithiol configuration with HS-SH distance of2.8 Å (Stourman et al. 2011), whereas ssYghU and ecYfcG each show adisulfide configuration with S—S distance of 2.1 Å (Wadington et al.2009, Patskovsky et al.).

In NaGST_(Nu), seven residues make close contacts with GSH1 (Thr51,Asn53, Gln86, Lys99, Ile100, Glu116, and Ser117) and three residues makeclose contacts with GSH2 (Asn25, Asn53, and Arg177 from the oppositechain in the dimer) (FIG. 8 (B)). These residues and contacts to theGSHs are conserved in the YghU structures ((Stourman et al. 2011,Patskovsky et al.); ecYfcG is missing an analogue of Asn25, as it istruncated at its N-terminus relative to the other proteins (FIG. 5). Theconserved threonine forms a hydrogen-bond with the GSH1 thiol that ischaracteristic of Nu-class GSTs (Stourman et al. 2011) (FIG. 8 (B); 3.0Å interatomic distance for Thr 51 in NaGST_(Nu)). ecYghU is predicted tohave one additional interaction with GSH1 (via Gln151 (Stourman et al.2011)) not present in NaGST_(Nu), ssYghU, or ecYfcG (which containVal150, Thr148, and Val105 at this position, respectively).

Alignment of the individual subunits of NaGST_(Nu), ecYghU and ssYghUshows four different configurations for their C-termini (FIG. 9 (A)):two for NaGST_(Nu) (FIG. 9 (B,C)) and one each for ecYghU (FIG. 9 (D))and ssYghU (FIG. 9 (E)). The C-terminal region of ecYfcG is inessentially the same configuration as that of ssYghU, though the lasteleven residues of ecYfcG are not present in its crystal structure. Thetwo NaGST_(Nu) subunits differ in the positioning of residues Leu258 toPhe288, with Phe288 being distant from the active site channel in onesubunit (5uun open, FIG. 9 (B)), and near the channel entrance in theother (5uun closed, FIG. 9 (C)), a difference of ˜18 Å. The closedNaGST_(Nu) configuration is stabilized by hydrogen-bonds between thesidechain amide of Lys262 and the carbonyl oxygen of Lys286, and thesidechain amide of Lys286 and the carbonyl oxygen of Arg220. The openconfiguration lacks these interactions and the sidechain of Arg220 hastwo rotamer positions. The sizes of the active site channel opening inthe open and closed configurations of NaGST_(Nu) are ˜18 Å² and ˜11 Å²,respectively (FIG. 9 (B,C)). For comparison, the area of active siteaccess for both ecYghU and ssYghU is ˜25 Å² (FIG. 9 (D,E)). Theplacement of helices-10 and 11 in ecYghU (FIG. 9 (D)), and the truncatedC-terminal sequence of ssYghU (FIG. 9 (D), FIG. 5), make it is unlikelythat either of these C-termini can occupy a position similar to thatseen in NaGST_(Nu).

Modeling of β(R)- and β(S)-GS-HPV into the GSH2 position in theNaGST_(Nu) active site predicts that their HPV moieties extend into theactive site channel in different orientations (FIG. 10 (A)). The channelinterior includes the phenol groups of Tyr166 and Tyr224, and thecarboxylate of Phe288 in the closed configuration. Our models predictthat these three residues interact differently with β(R)- andβ(S)-GS-HPV. With the less reactive β(R)-GS-HPV, Tyr166 is predicted toprovide a long interaction (4 Å) with the α-ketone, whereas Tyr224hydrogen-bonds to the γ-hydroxyl (3.1 Å), which in turn hydrogen-bondsto the α-ketone (FIG. 10 (B)). With the more reactive β(S)-GS-HPV,Tyr166 is predicted to provide a cation-n interaction with its aromaticring (average distance 3.4 Å), whereas Tyr224 provides a hydrogen-bondto its γ-hydroxyl (FIG. 10 (C)). Phe288 is also predicted tohydrogen-bond with the phenolic group of both β(R)- and β(S)-GS-HPV inthe closed configuration of NaGST_(Nu).

Discussion

In developing bio-based systems to depolymerize lignin, optimizedcellular and enzyme catalysts are needed. In this study, we testedsphingomonads for the ability to break the β-aryl ether bond commonlyfound in lignin (FIG. 1), and characterized a Nu-classglutathione-S-transferase from N. aromaticivorans that acts as aglutathione lyase in the β-etherase pathway (NaGST_(Nu)). Our findingthat other Nu-class GSTs also catalyze this reaction provides importantinsight into the function of this enzyme family in lignindepolymerization and possibly metabolism of other compounds that proceedvia GSH-conjugates.

Differences in GGE Metabolism.

We found that N. aromaticivorans was the most effective species studiedhere at metabolizing the dimeric aromatic compound GGE and assimilatingit into cellular material. The rate of GGE metabolism by N.aromaticivorans is comparable to those of Erythrobacter sp. SG61-1L(Palamuru et al. 2015) and Novosphingobium sp. MBESO4 (Ohta et al. 2015)(FIG. 3). Sphingobium sp. SYK-6 (Palamuru et al. 2015), Novosphingobiumsp. PP1Y (FIG. 4A), and S. xenophagum (FIG. 4B) are slower and/or lessefficient at metabolizing GGE, even though they each contain enzymesimplicated in the β-etherase pathway.

GGE Metabolism by N. aromaticivorans.

The appearance of extracellular MPHPV, threo-GGE, and HPV in N.aromaticivorans cultures suggests that it excretes these β-etherasepathway intermediates (FIGS. 2A and 2B). The appearance of threo-GGE incultures fed erythro-GGE shows that GGE oxidation is reversible in vivo,as was previously found for Pseudomonas acidovorans D3 (Vicuña et al.1987). The low level of extracellular guaiacol, and the absence ofextracellular GS-HPV in GGE-fed cultures suggest that MPHPV cleavageoccurred intracellularly, as was proposed for Novosphingobium sp. MBESO4(Ohta et al. 2015), and which was expected, based on the requirement forGSH for this reaction (FIG. 1). Since the conversion of GS-HPV into HPValso requires GSH, this reaction also likely occurred intracellularly.The relatively late uptake of HPV from the media (FIGS. 2A and 2B(panels B,H)) suggests that N. aromaticivorans metabolized andassimilated guaiacol before HPV. In contrast, Erythrobacter sp. SG61-1Lmetabolized HPV, but not guaiacol (Palamura et al. 2015), andPseudomonas acidovorans E-3 consumed the guaiacol only after consumingthe phenylpropanoid formed from splitting veratrylglycerol-β-guaiacylether (Crawford et al. 1973). This shows that species use differentstrategies for metabolizing β-etherase pathway intermediates, a featurethat could be useful in developing strains that produce specific pathwayintermediates.

Bacterial metabolism of HPV has been proposed to proceed throughacetovanillone, vanillin, vanillate, and protocatechuate (Crawford etal., 1973; Masai et al., 2007; Palamuru et al., 2015; Vicuña et al.,1987), and metabolism of guaiacol has been proposed to proceed throughcatechol (Crawford et al., 1973). However, we failed to detect any ofthese compounds in N. aromaticivorans culture media, suggesting thataromatic intermediates downstream of HPV and guaiacol were retainedwithin the cells upon formation.

Nu-Class GSTs can Function as Glutathione Lyases.

We found that NaGST_(Nu), SYK6GST_(Nu), ecYghU, and ecYfcG cleave theGS-moiety from both β(R)- and β(S)-GS-HPV, though with a wide range ofcatalytic efficiencies (k_(cat)/K_(M)) (at least 10⁴-fold; Table 7).Thus, along with GST3 from Novosphingobium sp. MBESO4 (Ohta et al.,2015), all five of the Nu-class GSTs that have been tested for GS-HPVcleavage show glutathione lyase (deglutathionylation) activity with bothstereoisomers of this substrate. Phylogenetic analysis of Nu-class GSTsshows these enzymes lie in widely separate sub-clades, suggesting thatthis activity may be widespread throughout this large class of proteins(FIG. 13).

Proposed Mechanism for the Nu-Class GST Glutathione Lyase Reaction.

We modeled the GS-moiety of GS-HPV into the GSH2 active site position ofNaGST_(Nu) (FIG. 8 (B)), with the HPV moieties of β(R)- and β(S)-GS-HPVextending into the active site channel in different orientations (FIG.10(A) and FIG. 12 (A,B)). We propose a mechanism for the glutathionelyase reaction (FIG. 11) in which the thiol of the GSH1 molecule (FIG. 8(B)) is activated by hydrogen-bonding with the conserved active sitethreonine (Thr51), which has moderate reactivity as a base at pH 8.5(FIG. 11 (A)). The activated GS1 thiol attacks the GS- of GS-HPV to forma disulfide GS-SG. In our proposed mechanism, the lyase reaction (C—Sbond cleavage) is facilitated by polarization of the GS-HPV α-ketone byinteractions involving Tyr166 and Tyr224 (which are highly conservedamongst many Nu-class GSTs; FIG. 5), and the γ-hydroxyl of HPV, withdifferent interactions for the two GS-HPV stereoisomers (FIG. 11 (A)).With β(R)-GS-HPV, Tyr166 is predicted to provide a long interaction (3.9Å) with the α-ketone, while Tyr224 hydrogen-bonds to the γ-hydroxyl (3.1Å), which in turn hydrogen-bonds to the α-ketone (FIGS. 10 (B) and 11(A)). Tyr166 is predicted to provide a cation-π interaction with thearomatic ring of the tighter binding β(S)-GS-HPV (average distance 3.4Å), while Tyr224 provides a hydrogen-bond to its γ-hydroxyl (FIGS. 10(C) and 11 (A)). Phe288 is also predicted to hydrogen-bond with thephenolic group of both β(R)- and β(S)-GS-HPV in the closed C-terminalconfiguration of NaGST_(Nu) (FIGS. 10 (B,C) and 11). ecYfcG lacksanalogues of these Tyr residues (FIG. 5), which may contribute to itsdiminished catalytic capability (k_(cat)/K_(M) values ˜10⁴-fold lowerthan those of NaGST_(Nu); Table 7). In the absence of a redox cofactor,we propose that a transient enolate (FIG. 11 (B)) stores the 2e⁻reducing equivalents released by disulfide bond formation as anincipient carbanion. Due to active site steric constraints, our modelplaces the reactive portion (S—C_(β)—(C_(α)═O)-aryl) of both β(R)- andβ(S)-GS-HPV into roughly planar configurations in the NaGST_(Nu) channel(FIG. 10 and FIG. 12 (A,B)), which should promote the formation of theenolate intermediate (FIG. 11 (B)). In contrast, steric constraints inthe ecYghU active site channel resulting from differences between itschannel interior and that of NaGST_(Nu) place these reactive GS-HPVatoms ˜45° out of alignment in our ecYghU models (FIG. 12 (C,D)),providing a possible explanation for the slower reactivity of ecYghUwith GS-HPV compared to NaGST_(Nu) (ecYghU has values of k_(cat)/K_(M)˜100-fold lower than those of NaGST_(Nu); Table 7). Collapse of theproposed enolate intermediate proceeds with carbanion trapping of asolvent-derived proton, corresponding to reduction of the carbon atomoriginally containing the thioether bond (FIG. 11 (C)).

This proposed mechanism for Nu-class GSTs with GS-HPV is different fromthat proposed for the omega class GST LigG, in which β(R)-GS-HPVinitially forms a mixed disulfide with a cysteine residue, releasing theHPV moiety (Pereira et al. 2016). A GSH molecule then enters the LigGactive site and combines with the enzyme bound GS-moiety to form GSSG. Aside chain thiol is unlikely to be involved in Nu-class glutathionelyase activity, since NaGST_(Nu) only contains one cysteine, which is˜21 Å away from the active site, and SYK6GST_(Nu) and ecYghU do notcontain any cysteine residues.

NaGST_(Nu) Converts β(R)- and β(S)-GS-HPV into HPV in N.aromaticivorans.

Although GST3 from Novosphingobium sp. MBESO4 can convert β(R)- andβ(S)-GS-HPV into HPV in vitro (Ohta et al. 2015), a physiological rolein the β-etherase pathway has not been established. The inability of N.aromaticivorans 12444Δ2595 to completely metabolize GGE (FIGS. 2A and 2B(panels C,D,I,J)) shows that NaGST_(Nu) is necessary for the β-etherasepathway in N. aromaticivorans. Unexpectedly, we found that 12444Δ2595accumulated extracellular MPHPV. Cleavage of MPHPV into guaiacol andGS-HPV is catalyzed by LigF and LigE/P (FIG. 1), enzymes that are likelyexpressed in 12444Δ2595, since crude extract from this strain can cleaveMPHPV (FIG. 7 (B)). We hypothesize that without NaGST_(Nu), GS-HPVaccumulates intracellularly in 12444Δ2595, and cells become limited forthe GSH that is needed to cleave MPHPV.

It is unclear whether the trace amount of HPV formed in assays using12444Δ2595 extract (FIG. 7 (B)) resulted from activity of an unknownenzyme or spontaneous cleavage of GS-HPV. In any event, addition ofrecombinant NaGST_(Nu) to the 12444Δ2595 extract resulted in thecomplete conversion of GS-HPV into HPV (FIG. 7 (B)), providing the firstdemonstration of a single enzyme being sufficient for one of the stepsof the β-etherase pathway in vivo.

The Role of Nu-Class GSTs in the β-Etherase Pathway.

The ability of Nu-class GSTs to cleave both β(R)- and β(S)-GS-HPV raisesthe question of why some species contain both this enzyme and LigG (e.g.Sphingobium sp. SYK-6), which is specific for the β(R)-isomer (FIG. 1).Our data show that both NaGST_(Nu) and SYK6GST_(Nu) have a higherk_(cat)/K_(M) value with β(S)-GS-HPV than with the β(R)-isomer (Table7). For NaGST_(Nu), this difference in k_(cat)/K_(M) values between thestereoisomers was ˜4-fold and the values (˜8000 and ˜1900 mM⁻¹ s⁻¹ forthe β(S)- and β(R)-isomers, respectively) are both greater than thatreported for LigG with β(R)-GS-HPV (1700 mM⁻¹ s⁻¹ (Pereira et al.2016)). For SYK6GST_(Nu), the difference in k_(cat)/K_(M) values betweenthe isomers is ˜10-fold, and the value with β(R)-GS-HPV (˜240 mM⁻¹ s⁻¹)is lower than that reported for LigG. These observations suggest thatSYK6GST_(Nu) likely cleaves β(S)-GS-HPV in Sphingobium sp. SYK-6, butthat LigG may play a role in cleaving β(R)-GS-HPV in that organism.Although cell extract from a Sphingobium sp. SYK-6 ΔLigG mutantcompletely cleaved a racemic GS-HPV sample (since the lysate presumablycontained active SYK6GST_(Nu)) the physiological effect of deleting LigGwas not reported (Masai et al. 2003). Perhaps organisms like N.aromaticivorans that lack LigG need Nu-class GSTs to have higherefficiencies toward β(R)-GS-HPV than Nu-class GSTs in species thatcontain a stereospecific enzyme like LigG.

The Potential Role(s) of Nu-Class GSTs in Bacteria that do not Containthe 13-Etherase Pathway.

Many organisms contain Nu-class GSTs, including those not known orpredicted to use the β-etherase pathway (Mashiyama et al., 2014;Stourman et al., 2011). Whereas several of these enzymes have been foundto have disulfide bond reductase activity in vitro, the physiologicalroles of most of these proteins are unknown (Mashiyama et al., 2014;Stourman et al., 2011). We found that ecYghU and ecYfcG from E. coli, anorganism not known to metabolize lignin-derived phenylpropanoids, cancleave both β(R)- and β(S)-GS-HPV in vitro, though with lower catalyticefficiencies than NaGST_(Nu) and SYK6GST_(Nu) (Table 2). The fact thatecYghU can replace NaGST_(Nu) in N. aromaticivorans (FIGS. 2A and 2B(panels E,F,K,L)) shows that it can indeed function as a glutathionelyase in vivo. The relatively low k_(cat)/K_(M) values of ecYghU andecYfcG in cleavage of GS-HPV compared to NaGST_(Nu) and SYK6GST_(Nu)could reflect the fact that GS-HPV is not a natural substrate for the E.coli enzymes. Although the overall structures of Nu-class GSTs aresimilar, differences in the residues surrounding the active sites (asseen between NaGST_(Nu) and ecYghU, for example; FIG. 12) could makeenzymes from other organisms better suited for binding and cleavingother GS-conjugates that they more commonly encounter.

Conclusion

This work shows that N. aromaticivorans can rapidly and completelymetabolize the β-aryl ether-containing compound GGE, and that theNu-class glutathione-S-transferase NaGST_(Nu) plays a direct role inthis process. The following example illustrates that NaGST_(Nu) canparticipate in cleavage of bona fide lignin oligomers in vitro,indicating utility of this enzyme in converting biomass into valuablechemicals. NaGST_(Nu) and other Nu-class GSTs can cleave both the β(R)-and β(S)-stereoisomers of the β-etherase pathway intermediate GS-HPV, incontrast to the other characterized enzymes in the pathway, which arestereospecific (FIG. 1). Our finding that ecYghU also cleaves GS-HPVshows that Nu-class GSTs from organisms lacking the β-etherase pathwaycan nevertheless act as racemic glutathione lyases.

Example 2

In Vitro Enzymatic Release of Syringyl, Guaiacyl, and Tricin Units fromLignin

Summary

New information and processes are needed to derive valuable compoundsfrom renewable resources. Lignin is an abundant, heterogeneous, andracemic polymer in terrestrial plants, and it is comprised predominantlyof guaiacyl and syringyl monoaromatic phenylpropanoid units that arecovalently linked together in a purely chemical radical couplingpolymerization process. In addition, the plant secondary metabolite,tricin, is a recently found and abundant lignin monomer in grasses. Themost prevalent type of inter-unit linkage between guaiacyl, syringyl,and tricin units is the β-ether linkage. Previous studies have shownthat enzymes in the bacterial β-etherase pathway catalyzeglutathione-dependent cleavage of β-ether bonds in dimeric β-etherlignin model compounds, resulting in the release of monoaromaticproducts, the reduction of nicotinamide adenine dinucleotide (NAD⁺) toNADH, and the oxidation of glutathione (GSH) to glutathione disulfide(GSSG). To date, however, it remains unclear whether the knownβ-etherase enzymes are active on lignin polymers. Here, we report onenzymes that catalyze β-ether cleavage from model compounds and bonafide lignin, under conditions that recycle the cosubstrates NAD⁺ andGSH. Guaiacyl, syringyl and tricin derivatives were identified asreaction products when different model compounds or lignin fractionswere used as substrates. These results provide the first demonstrationof an in vitro enzymatic system that can recycle NAD⁺ and GSH whilereleasing aromatic monomers from model compounds as well as natural andengineered lignin oligomers. These findings can improve the ability toproduce valuable aromatic compounds from a renewable resource likelignin.

Introduction

There is economic and environmental interest in using renewableresources as raw materials for production of chemicals that arecurrently derived from fossil fuels. Lignin, a renewable resource thataccounts for ˜15-30% (dry weight) of vascular plant cell walls (Higuchi1980, Lewis et al. 1990), is comprised of aromatic compounds that may bevaluable commodities for the biofuel, chemical, cosmetic, food, andpharmaceutical industries (Sinha et al. 2008). Consequently, intensiveefforts are currently aimed at developing chemical, enzymatic and hybridmethods for deriving simpler and lower molecular weight products fromlignin (Gall et al. 2017).

The lignin backbone is predominantly composed of guaiacyl (G) andsyringyl (S) phenylpropanoid units (FIG. 16 (A)) that derive from themonomers coniferyl and sinapyl alcohol that become covalently linkedduring lignification via radical coupling reactions, primarily byendwise addition of a monomer (radical) to the phenolic end of thegrowing polymer (radical). G and S units are inter-linked by a varietyof chemical bonds by which the units are characterized: resinols (β-β),4-O-5-diaryl ethers, phenylcoumarans (β-5), and β-O-4-aryl ethers(termed β-ethers hereafter)] (Adler 1977, Adler 1957, Adler 1955). Ingrasses, the flavone tricin (T units, FIG. 16 (A)) begins a chain and iscovalently linked to the next unit via a 4-O-β-ether bond (Lan et al.2016, Lan et al. 2015, del Rio et al. 2012). Given that approximately50-70% of all inter-unit linkages in lignin are β-ethers (Adler 1977,Adler 1957, Adler 1955), cleavage of these bonds is crucial forprocesses aiming to derive valuable low molecular weight compounds fromlignin in high yields. The formation of β-ether linkages duringlignification generates a racemic lignin product containing both β(R)-and β(S)-carbons that, after re-aromatization of the quinone methideintermediate by proton-assisted water addition, are adjacent to eitherα(R)- or α(S)-configured benzylic alcohols (Akiyama et al. 2002,Sugimoto et al. 2002, Ralph et al. 1999). Each unit therefore has 4optical isomers and two ‘real’ isomers—the so-designated threo anderythro (or syn and anti) isomers. Lignin depolymerization via β-etherbond cleavage has been demonstrated with chemical catalysis (Rahimi etal. 2013, Rahimi et al. 2014). In addition, cytoplasmic enzymes in asphingomonad β-etherase pathway have been identified that oxidize andcleave model β-ether linked aromatic dimers (Masai et al. 2007).

The β-etherase pathway is present in Sphingobium sp. strain SYK-6 andother sphingomonads (e.g., Novosphingobium spp.) (Masai et al. 2007,Gall and Ralph et al. 2014). The diaromatic β-ether-linkedguaiacylglycerol-β-guaiacyl ether (GGE, FIG. 16 (B)) lignin modelcompound has been used as a substrate to identify the following threeenzymatic steps in cleavage of β-ether linkages in vitro (Gall and Ralphet al. 2014, Masai et al. 2003, Sato et al. 2009, Tanamura et al. 2010,Gall and Kim et al. 2014): (1) a set of dehydrogenases catalyzenicotinamide adenine dinucleotide (NAD⁺)-dependent α-oxidation of GGE toGGE-ketone and NADH (Sato et al. 2009, Masai and Kubota et al. 1993),(2) β-etherases that are members of the glutathione S-transferasesuperfamily, carry out glutathione (GSH)-dependent cleavage ofGGE-ketone, releasing guaiacol andβ-S-glutathionyl-γ-hydroxy-propiovanillone (GS-HPV) (Masai et al. 2003,Gall and Kim et al. 2014, Masai and Katayama et al. 1993), and (3) oneor more glutathione lyases catalyze GSH-dependent cleavage of GS-HPV,yielding glutathione disulfide (GSSG) and γ-hydroxypropiovanillone (HPV)(Masai et al. 2003, Gall and Kim et al. 2014, Rosini et al. 2016, Konturet al. 2018).

The use for multiple enzymes at some of the pathway's steps isattributable to the existence of both R- and S-configured chiral centersin lignin (Akiyama et al. 2002, Sugimoto et al. 2002, Ralph et al.1999). The known NAD⁺-dependent dehydrogenases (LigD, LigL, LigN, andLigO) exhibit strict stereospecificity at the a position withindifference to the configuration at the β position (Sato et al. 2009).With model diaromatic substrates, LigD and LigO are active on theR-configured α-epimers, whereas LigL and LigN are active on theS-configured α-epimers. Because the combined activity of thesedehydrogenases eliminates the chiral center at a, GGE-ketone exists astwo β-enantiomers that are cleaved by stereospecific β-etherases LigE,LigP and LigF, each of which catalyzes the release of guaiacol withchiral inversion at the β position, and one of two β-epimers of GS-HPV(LigE and LigP convert β(R)-GGE-ketone to β(S)-GS-HPV and LigF convertsβ(S)-GGE-ketone to β(R)-GS-HPV) (Gall and Kim et al. 2014). The finalstep is the GSH-dependent cleavage of the GS-HPV epimers, yielding GSSGand HPV as coproducts. LigG has been shown to cleave both β(R)-GS-HPVand β(S)-GS-HPV (Rosini et al. 2016), although it appears to have astrong preference for the former (Masai et al. 2003, Gall and Kim et al.2014). Recently, a GSH transferase from Novosphingobium aromaticivoransDSM12444 (NaGST_(NU); Saro_2595 in GenBank assembly GCA_000013325.1)(Kontur et al. 2018) has been shown to have high activity withβ(R)-GS-HPV and β(S)-GS-HPV both in vivo and in vitro, producing HPV andGSSG as products (FIG. 16 (B)).

Despite what is known about the activity of individual β-etherasepathway enzymes with model diaromatic compounds, there is littleinformation on their function with lignin oligomers. In vivo activitymay be limited to aromatic dimers or small lignin oligomers due torestrictions in transporting large polymers into the bacterial cytoplasmwhere the β-etherase pathway enzymes are found. To better understand thefunction of β-etherase pathway enzymes, we sought to use a minimal setof enzymes to develop a coupled in vitro assay capable of releasing G, Sand T aromatic monomers and recycling the cosubstrates NAD⁺ and GSH.Here we demonstrate complete conversion of GGE to guaiacol and HPV in areaction containing LigD, LigN, LigE, LigF, NaGST_(Nu), and theAllochromatium vinosum DSM180 GSH reductase (AvGR), which catalyzesNADH-dependent reduction of GSSG (FIG. 16 (B)) (Reiter et al. 2013). Wealso show that this same combination of enzymes releases tricin from themodel compound guaiacylglycerol-β-tricin ether (GTE, FIG. 16 (C)). Inaddition, we show that the same combination of enzymes releases G, S,and T units from bona fide lignin oligomers; this is the first report todemonstrate the release of tricin from lignin units by biologicalmethods. We discuss new insights gained from this study and itsimplications for the future production of these and possibly othervaluable products from lignin.

Methods

General.

GGE was purchased from TCI America (Portland, Oreg.). Tricin, GTE,GTE-ketone, HPV, γ-hydroxypropiosyringone (HPS) and GGE-ketone weresynthesized by previously described methods (Adler et al. 1955, Lan etal. 2015, Masai et al. 1989). All other chemicals were purchased fromSigma-Aldrich (St. Louis, Mo.). Methods to isolate and characterizemaize (Zea mays) corn stover (MCS) and hybrid poplar (HP) lignin sampleswere described previously (Lan et al. 2015, Stewart et al. 2009, Shuaiet al. 2016). ¹H and ¹³C NMR spectra were recorded on a Bruker Biospin(Billerica, Mass.) AVANCE 700 MHz spectrometer fitted with acryogenically-cooled 5-mm quadruple-resonance ¹H/³¹P/¹³C/¹⁵N QCIgradient probe with inverse geometry (proton coils closest to thesample). Manipulation of DNA and preparation of Escherichia colitransformant cultures were carried out according to previously describedmethods (Moore 2003). All lig genes from Sphingobium sp. strain SYK-6,as well as those encoding AvGR from A. vinosum DSM180 were codonoptimized for expression in E. coli and obtained from GeneArt® (LifeTechnologies). NaGST_(Nu) was amplified and cloned from N.aromaticivorans DSM12444 genomic DNA (Kontur et al. 2018).

Plasmid and Protein Preparation.

Procedures for cloning, recombinant expression and purification of Tevprotease, LigE, LigF, LigG, and NaGST_(Nu) are described elsewhere (Galland Kim et al. 2014, Kontur et al. 2017). Codon-optimized ligD, ligN andgenes AvGR were cloned into plasmid pVP302K (Gall and Kim et al. 2014)via the PCR overlap method (Shevchuk et al. 2004, Bryksin et al. 2010,Horton et al. 2013, Horton 1993). Expression and purification of LigD,LigN, NaGST_(Nu), and AvGR followed similar procedures as those usedpreviously (Gall and Kim et al. 2014). Briefly, E. coli strain B834cultures, transformed with expression plasmids, were grown aerobicallyovernight in 1 L of auto-induction ZYM-5052 medium (Studier 2005)supplemented with 100 μg mL⁻¹ kanamycin. Cells were pelleted andextracts prepared via compression and sonication. Histidine-taggedproteins were purified from cell lysates via Ni-NTA affinitychromatography with QIAGEN Ni-NTA resin. His-tagged Tev protease wasused to liberate N-terminal His-tags and a second round of Ni-NTAaffinity chromatography was used to remove the tag and Tev proteasebefore separation by size-exclusion chromatography. Protein preparationswere concentrated and frozen with liquid N₂.

Enzyme Assays.

In vitro enzyme assays containing LigD, LigN, LigE, LigF, NaGST_(Nu) (orLigG), and AvGR (or a subset of those enzymes) were conducted in assaybuffer (25 mM Tris, 2.0% DMSO, pH 8.0). The concentration of each enzymewas 50 μg mL⁻¹ in all assays. When GGE (6 mM) was the substrate, theinitial cosubstrate concentrations were 2 mM NAD⁺ and 4 mM GSH. When GTE(1 mM) was the substrate, the initial cosubstrate concentrations were 5mM NAD⁺ and 5 mM GSH. When an isolated lignin sample was used as thesubstrate (2.2 mg mL⁻¹), the initial cosubstrate concentrations were 2mM NAD⁺ and 4 mM GSH. Enzyme assays (1 mL or larger volume as needed)were carried out (in duplicate) as follows: (1) the substrate (GGE, GTE,or lignin) was dissolved in DMSO (50-times concentrated above theintended assay concentration) and 20 μL of the solution were added to a2 mL vial, (2) 880 μL of 25.6 mM Tris pH 11.5 (where the acidic effectof GSH drops the pH to 8.0 after addition of 5 mM GSH), (3) 50 μL of astock solution in 25 mM Tris containing NAD⁺ and GSH (each is 20-timesconcentrated above the intended assay concentration), and (4) 50 μL of20-times concentrated mixture of the desired enzymes. At indicated timepoints, 150 μL samples were removed from an assay and enzymatic activitywas abolished by pipetting into 5 μL of 5 M phosphoric acid. GGE,guaiacol, HPV, and HPS concentrations were quantified for each timepoint [see below] using a linear regression of known standards for eachcompound.

Preparative Gel-Permeation Chromatography (GPC).

GPC of lignin samples was carried out using a Beckman 125NM solventdelivery module equipped with a Beckman 168 UV detector (λ=280 nm) and a30 mL Bio-Rad Bio Bead S-X3 column (a neutral, porousstyrene-divinylbenzene copolymer). Dimethylformamide (DMF) was used asthe mobile phase at a flow rate of 1.0 mL min⁻¹. Between 20 and 50 mg oflignin was dissolved in a minimal amount of DMF, injected into themobile phase, and 1 mL fractions were collected until UV absorptiondecreased to baseline levels. Fractions were then subjected toanalytical GPC to estimate their average molecular weight (MW). The DMFwas evaporated in vacuo in order to recover material used for enzymeassays.

Analytical GPC.

Analytical GPC of lignin samples was carried out with a ShimadzuProminence Ultra Fast Liquid Chromatography system (LC-20AD pumps,SIL-20AC HT autosampler, CTO-20A column oven and CBM-20A controller) andusing two TSKgel Alpha-2500 (300×7.8 mm; Tosoh Bioscience) columns at40° C. Samples (10 μL injection volume) containing approximately 1 mgmL⁻¹ of isolated or GPC-fractionated lignin were injected into a mobilephase (100 μM LiBr in DMF) at a flow rate of 0.3 mL min⁻¹ with a runlength of 90 min. An SPD-M20A photodiode array detector (λ=200 nm) wasused for the determination of elution times that were subsequentlyconverted to MW values using regression analysis of ReadyCal-KitPolystyrene standards.

C₁₈-Chromatography.

C₁₈-Chromatographic separations were carried out using a Beckman 125NMsolvent delivery module equipped with a Beckman 168 UV detector. 150 μLsamples from enzyme assays were collected and 20 μL aliquots wereinjected into either a 4×120 mm Restek Ultra Aqueous C₁₈-reversedstationary phase column, or a 4.6×250 mm Phenomenex Luna 5uC₁₈(2)-reversed stationary phase column with a 1.0 mL min⁻¹ mobile phasecomposed of a mixture of an aqueous buffer (5 mM formic acid in 95/5H₂O/acetonitrile) and methanol. Samples from enzyme assays using GTE asthe substrate were analyzed on the Phenomenex column to improveseparation of GTE and tricin. All other C₁₈-chromatographic separationswere carried out using the Restek column. For the Restek column, themethanol fraction of the buffer (with water as the remainder) wasadjusted as follows: 0-6 min, 30% methanol; 6-15 min, gradient from 30to 80% methanol; 15-27 min, 80% methanol; 27-28 min, gradient from 80 to30% methanol; 28-33 min, 30% methanol. For the Phenomenex column, thegradient system was as follows: 0-6 min, 10% methanol; 6-50 min,gradient from 10 to 90% methanol; 50-63 min, 90% methanol; 63-64 min,gradient from 90 to 10% methanol; 64-70 min, 10% methanol.

Results

Design of a Coupled In Vitro Assay for Cleavage of β-Ether-LinkedDiaromatic Compounds.

As an initial substrate for this assay we used erythro-GGE, which is amixture of enantiomers (αR,βS)-GGE and (αS,βR)-GGE that has been usedextensively as a substrate with β-etherase pathway enzymes vitro (Galland Ralph et al. 2014, Masai et al. 2003, Sato et al. 2009, Tanamura etal. 2010, Gall and Kim et al. 2014). We used recombinant preparations ofLigD and LigN as these dehydrogenases are reported to be sufficient forthe NAD⁺-dependent oxidation of R- and S-configured α-anomers oferythro-GGE in vitro (Sato et al. 2009). The assay also containedrecombinant preparations of LigE and LigF that have been shown toseparately catalyze the GSH-dependent conversion of a racemic mixture ofGGE to guaiacol and the S- and R-epimers of GSH-HPV (Gall and Kim et al.2014). NaGST_(Nu) was present to catalyze the GSH-dependent cleavage ofthe GSH-HPV epimers to HPV and GSSG. The properties of individualenzymes (FIG. 16 (B)) predicts that this coupled system will requireequimolar concentrations of GGE and NAD⁺ and twice as much GSH forcomplete conversion of GGE to HPV and guaiacol.

In an attempt to reduce the amount of added NAD⁺ and GSH that would beneeded for full conversion of diaromatic substrate to products, somereactions included recombinant AvGR, which catalyzes the NADH-dependentreduction of GSSG (Reiter et al. 2013), thereby recycling thecosubstrates NAD⁺ and GSH for continued conversion of the β-ethersubstrates. This cosubstrate recycling system was tested with 6 mMerythro-GGE and limiting concentrations of NAD⁺ (2 mM) and GSH (4 mM)(FIG. 17A (panel A)). Using a mixture of LigD, LigN, LigE, LigF, andNaGST_(Nu) (without AvGR), we observed that erythro-GGE was partiallyconverted to HPV and guaiacol (FIG. 17A (panel B)). Quantification ofthis assay revealed that the erythro-GGE concentration decreased from6.0 mM to 3.8 mM at the end of the assay, whereas the HPV and guaiacolconcentrations were each 2.0 mM, the NAD⁺ levels were non-detectable,and the threo-GGE [a mixture of enantiomers (αR,βR)-GGE and (αS,βS)-GGE]concentration increased to 0.1 mM presumably due to the reportedreversibility of the LigD/LigN reactions (Pereira et al. 2016). Thus,the final GGE concentration (3.9 mM, the sum of erythro-GGE andthreo-GGE concentrations) was consistent with consumption of 2.0 mMNAD⁺. In addition, the production of 2.0 mM (each) of HPV and guaiacolwas consistent with the consumption of 4.0 mM GSH, where 2.0 mM GSH wasconsumed in the LigE/LigF reactions and an additional 2.0 mM GSH wasconsumed in the NaGST_(Nu) reaction.

To test the impact of AvGR on this assay, we added it to a parallel invitro reaction. In the presence of AvGR (FIG. 17A (panel C)), we foundthat GGE was completely consumed along with the appearance of equimolaramounts of HPV and guaiacol (6.0 mM each) without a detectable change inthe NAD⁺ concentration or accumulation of any β-etherase pathwayintermediates by the time of the assay's conclusion. To determine if anyβ-etherase pathway intermediates accumulated over the course of theassay, we tested for time-dependent changes in the concentrations of thesubstrate, known pathway intermediates and products in a parallelreaction (FIG. 18). We found that as erythro-GGE degradation occursthere is a time-dependent accumulation and depletion of GGE-ketone andthreo-GGE and, eventually, complete equimolar conversion of thesubstrate to HPV and guaiacol (FIG. 17A (panels A-C)). From theseresults, we conclude that the combination of LigD, LigN, LigE, LigF,NaGST_(Nu), and AvGR is sufficient to process all of the chiral centersin a β-ether substrate such as erythro-GGE. In addition, we concludethat the presence of AvGR is sufficient to recycle the cosubstrates NAD⁺and GSH that are needed for cleavage of β-ether bonds in a modeldiaromatic compound such as erythro-GGE.

From information available in the literature, it has remained unclearwhether the GSH lyase from Sphingobium strain SYK-6, LigG, exhibits apreference for β(R)-GS-HPV (Masai et al. 2003, Gall and Kim et al.2014), or is capable of cleaving the thioether linkages in bothβ(R)-GS-HPV and β(S)-GS-HPV (Rosini et al. 2016). As the presence ofNaGST_(Nu) resulted in cleavage of both β(R)-GS-HPV and β(S)-GS-HPV inthis coupled reaction system (FIG. 17A (panels A-C)), we sought to usethis in vitro assay to test the activity of LigG under identicalconditions. When we performed an assay using 6.0 mM erythro-GGE, 2.0 mMNAD⁺, and 4.0 mM GSH, as well as the mixture of LigD, LigN, LigE, LigF,and LigG (without AvGR), we observed partial conversion of GGE to HPVand guaiacol (FIG. 17B (panel D)). At the end of this assay, the totalGGE concentration (4.0 mM, the sum of erythro-GGE and threo-GGEconcentrations) was expected based on the consumption of 2.0 mM NAD⁺.Further, the production of 2.0 mM (each) of HPV and guaiacol wasconsistent with the consumption of 4.0 mM GSH (2.0 mM GSH consumed byeach of the LigE/LigF and LigG reaction steps). When we added AvGR to aparallel reaction that contained GGE (6.0 mM), NAD⁺ (2 mM), GSH (4 mM)and a combination of LigD, LigN, LigE, LigF, and LigG, we did notobserve complete conversion of GGE to HPV and guaiacol (FIG. 17B (panelE)). Instead, we detected the diaromatic substrate (erythro-GGE),threo-GGE, GS-HPV, and GGE-ketone (0.7 mM). In contrast to what is foundwhen NaGST_(Nu) was present under identical reaction conditions, thepresence of LigG led to incomplete utilization of the diaromaticsubstrate and the accumulation of β-etherase pathway intermediates. Fromthese results we conclude that LigG is not able to completely cleaveboth β-epimers of GS-HPV in vitro. Consequently, all subsequent assayswere performed using NaGST_(Nu) as a source of GSH lyase activity.

Production of Tricin from GTE In Vitro.

In grasses, the flavone tricin (T, FIG. 16 (A)) is covalently linked toone end of lignin, via a β-ether bond (Lan et al. 2016, Lan et al. 2015,Lan et al. 2014). Although β-etherase pathway enzymes have been shown tocleave β-ether-linked diaromatic model compounds containing G and Smonomers, to date there is no published data on their ability to removethe diaromatic flavonoid T units from any substrate. Thus, we sought totest the ability of the coupled assay to cleave GTE (FIG. 16 (C)), amodel compound containing a β-ether linked tricin moiety. HPLC analysisof the synthetic GTE (FIG. 19A (A)) indicated that it contained a 6:1ratio of erythro-GTE [(αR,βS)-GTE and (αS,βR)-GTE] to threo-GTE[(αR,βR)-GTE and (αS,βS)-GTE], which was consistent with the NMRanalysis of this material (Lan et al. 2016).

When we incubated 1.0 mM GTE, 5.0 mM NAD⁺, 5.0 mM GSH with thecombination of LigD, LigN, LigE, LigF and NaGST_(Nu) (FIG. 19B (B)) weobserved the complete conversion of GTE to tricin and HPV. This resultpredicts that LigD and LigN oxidize GTE to form GTE-ketone, LigE andLigF catalyze β-ether cleavage in GTE-ketone to form GS-HPV and tricin,and NaGST_(Nu) releases HPV from GS-HPV (FIG. 16 (C)), suggesting thatthe larger β-ether-linked flavone model was able to access the activesites in LigD, LigN, LigE, and LigF. To further test this hypothesis, weassayed for the presence of the expected β-etherase pathwayintermediates, GS-HPV and GTE-ketone, from GTE. By performing a parallelreaction containing the same substrates and only LigD, LigN, LigE, andLigF (FIG. 19B (C)), we observed that GTE was degraded and tricin wasproduced. However, in this assay, there was no detectable production ofHPV and we observed accumulation of GS-HPV. These findings indicate thatthe absence of NaGST_(Nu) prevented the conversion of GS-HPV to HPV(FIG. 16 (C)). Finally, in an assay containing only the enzymes LigD andLigN (FIG. 19B (D)), we found that GTE was almost completely convertedto GTE-ketone, as expected from the NAD⁺-dependent α-oxidation activityof GTE. Together, the data show for the first time that T units can bederived from β-ether-linked model compounds in vitro using enzymes,cosubstrates and intermediates that are known to be part of theβ-etherase pathway (FIG. 16).

Release of G, S, and T Units from Lignin Oligomers.

With the coupled enzymatic system in place, we tested it for activitywith lignin oligomers. First, we tested if a mixture of LigD, LigN,LigE, LigF, NaGST_(Nu), and AvGR produced S units from a high-syringylhybrid poplar (HP) lignin polymer (Stewart et al. 2009, Shuai et al.2016). To ensure that the test was performed with lignin oligomersrather than low-MW material, we fractionated the HP lignin by GPC andpooled the high-MW fractions (FIG. 20, Table 9) for use as a substrate(FIG. 21 (A-B)). From 2.2 mg mL⁻¹ lignin oligomers having MW between9,000 and 12,000 (with 2 mM NAD⁺ and 4 mM GSH), we detected theproduction of 1.0 mM HPS, the HPV analog expected to be produced bycleavage of β-ether bonds from a syringyl unit at one end of the ligninchain. We also detected the formation of an unknown product in thisreaction (FIG. 21 (B)), which could be either a chemically modified Sunit released from the HP lignin or a GS-linked intermediate product.Furthermore, syringaresinol, a dimeric unit in the HP lignin polymer(Stewart et al. 2009), was not detected as a product of the enzymaticreaction.

TABLE 9 Estimated size of the HP lignin fractions after preparative GPC(size distributions are shown in FIG. 9-2). Size was determined fromanalytical GPC and is reported in Da and the corresponding polymerlength is reported in number of units, based on the MW of syringaresinol(418.44) and β-ether-linked syringyl units (228.24). Average Average MWLength (Units) Original sample, pre-fractionation 8,665 38.3 Fraction 1*11,550 51.0 Fraction 2* 10,780 47.6 Fraction 3* 9,340 41.3 Fraction 47,240 32.0 Fraction 5 5,200 23.0 Fraction 6 3,720 16.5 Fraction 7 2,66011.8 Fraction 8 1,910 8.5 Fraction 9 1,280 5.8 Asterisks highlightfractions that were pooled and used as the substrate in enzyme assays.

Given the ability of the enzymatic assay to release HPS from HP lignin,we also tested for the release of aromatic monomers from a more complexlignin, such as the one derived from maize corn stover (MCS) (Lan et al.2016, Lan et al. 2015). To generate substrates for these assays, we usedpreparative GPC to size-fractionate MCS lignin (FIG. 22, Table 10) andtested materials with different apparent MW values as the source oflignin oligomer substrates for enzyme assays (FIGS. 23A and 23B). Totest for activity with these samples, we incubated LigD, LigN, LigE,LigF, NaGST_(Nu), and AvGR with MCS lignin oligomers (2.2 mg mL⁻¹), 2.0mM NAD⁺ and 4.0 mM GSH (FIG. 23A (panel A)). In these experiments, wedetected release of HPV and HPS in assays using lignin oligomers withaverage MW ranging from 460 to 10,710 (FIG. 22). The highestconcentrations of HPV (0.4 mM) and HPS (0.1 mM) were observed withlignin oligomers having an average MW of 1,390 (FIG. 23B (panel D)) asthe substrate. In general, larger lignin oligomers resulted in loweraccumulation of HPV and HPS. In addition, similar to the observationswith HP lignin, unknown products were detected in most of the enzymaticreactions with the different lignin fractions (FIGS. 23A and 23B).Tricin was only observed as a reaction product when using the lowest MWfraction tested (MW=460, FIG. 23B (panel F)). In sum, we conclude fromthese experiments that a combination of LigD, LigN, LigE, LigF,NaGST_(Nu), and AvGR can release some, but not all, G, S and T unitsfrom MCS lignin oligomers.

TABLE 10 Estimate size of the MCS lignin fractions after preparative GPC(size distributions are shown in FIG. 5). Size was determined fromanalytical GPC and is reported in Da and the corresponding polymerlength is reported in number of units, based on the crude assumptionthat the average unit has a MW of 210. Average Average MW Length (Units)Original sample, pre-fractionation 5,980 28.5 Fraction 1* 10,710 51.0Fraction 2 9,860 46.9 Fraction 3 8,320 39.6 Fraction 4 6,690 31.9Fraction 5* 5,370 25.6 Fraction 6 3,930 18.7 Fraction 7 2,110 10.1Fraction 8* 1,390 6.6 Fraction 11 880 4.2 Fraction 14* 660 3.1 Fraction17* 460 2.2 Asterisks highlight fractions that were used as thesubstrate in enzyme assays.

Discussion

In order to use a polymer like lignin as a source of valuable aromaticsand other chemicals it is necessary to develop new or improve onexisting depolymerization strategies. There has been considerableinterest in exploring the use of the bacterial β-etherase pathway forthe biological production of aromatics from this renewable plantpolymer. There is now a large amount of information on the types ofmodel diaromatic substrates recognized by individual β-etherase enzymesin vitro, the products of their activity, and their structural orfunctional relationships to other known enzymes (Pereira et al. 2016,Helmich et al. 2016). Despite this, information is lacking on theiractivity with lignin oligomers. In addition, as these are cytoplasmicenzymes, it is plausible that they evolved to break down β-ether linksonly in the smaller lignin oligomers that could be transported insidethe cells. In this work, we sought to develop a coupled in vitro systemcontaining a set of β-etherase pathway enzymes that was capable ofreleasing monoaromatic compounds when incubated with differentsubstrates. We reasoned that such a system would provide additionalinformation on the β-etherase enzymes and aid in studies aimed atdetermining the requirements for release of valuable aromatics from bonafide lignin oligomers.

In this study, we identified a minimum set of enzymes (LigD, LigN, LigE,LigF, NaGST_(Nu), and AvGR) that is capable of cleaving β-ether linkagesand completely converting model diaromatic compounds to aromaticmonomers. We further showed that this coupled in vitro assay system iscapable of stoichiometric production of monoaromatic products from modeldiaromatics in the presence of limiting amounts of the cosubstrates NAD⁺and GSH. The ability to recycle NAD⁺ and GSH reduces the need forexpensive cofactors and increases the future utility of a coupled enzymesystem for processing lignin oligomers in vitro. Finally, we showed thatthis coupled enzyme system has activity with fractionated ligninoligomers. Below we summarize the new information gained from using thisassay with widely used or new model β-ether linked substrates as well aslignin oligomers of different sizes.

Insights Gained from Using the Coupled Assay with Diaromatic Compounds.

Using GGE as a substrate, we demonstrated that the GSH reductase AvGR iscapable of recycling the cosubstrates NAD⁺ and GSH, enabling theβ-etherase enzymes to completely cleave GGE in the presence ofsub-stoichiometric amounts of these cofactors (FIG. 17A (panels A-C)).The A. vinosum DSM180 AvGR is well-suited for this purpose, as mostglutathione reductases described in the literature use NADPH instead ofNADH as an electron donor (Reiter et al. 2014). When AvGR was notpresent in an assay in which GGE concentrations were greater than thoseof NAD⁺ and GSH, there was incomplete hydrolysis of this diaromaticsubstrate, accumulation of β-etherase pathway intermediates, anddepletion of NAD⁺, as expected if the reaction was cofactor limited.

We were also able to detect the release of tricin when GTE was used as asubstrate in this assay, showing for the first time that β-etherasepathway enzymes are capable of β-ether bond cleavage in a substratebearing a large flavonoid moiety. This further shows that the β-etherasepathway enzymes are not limited to substrates containing only G and Smonoaromatic units. In prior research, we had demonstrated the abilityof LigE and LigF to cleave G-G, G-S, S-G, and S—S dimer models (Gall andRalph et al. 2014, Gall and Kim et al. 2014), so this result extends theknowledge of the diversity of substrates for these enzymes to the G-Tdimers. Thus, although the β-etherase pathway enzymes are thought to behighly stereospecific, they are also capable of recognizing the manydifferent configurations of β-ether linked aromatics potentially presentin lignin. With the results of these and previous findings combined(Gall and Ralph et al. 2014, Gall and Kim et al. 2014), we conclude thatthe minimal set of enzymes used in this study is sufficient to enablethe β-etherase pathway in vitro to release of G, S, and T units fromcompounds modeling β-ether units in lignin.

This coupled assay also allowed us to directly compare the ability ofLigG and NaGST_(Nu) to function in the β-etherase pathway. We found thatthe presence of NaGST_(Nu), AvGR, along with LigD, LigN, LigE, and LigF,was sufficient to allow complete conversion of GGE to HPV and guaiacol(FIG. 17A (panels A-C)). This is consistent with our prediction thatNaGST_(Nu) can accommodate both GS-HPV epimers in its active site(Kontur et al. 2018) and the ability of this enzyme to producestoichiometric amounts of HPV from GGE when added to this coupled assay.In contrast when LigG replaced NaGST_(Nu) under otherwise identicalassay conditions, there was incomplete hydrolysis of GGE to HPV andguaiacol, with significant accumulation of GGE-ketone and lower amountsof GS-HPV (FIGS. 17A and 17B (panels A,D-E)). Thus, although it has beensuggested that LigG can hydrolyze both β-epimers of GS-HPV (Rosini etal. 2016), this result, along with those published previously (Gall andKim et al. 2014), support the hypothesis that LigG has a strongpreference for β(R)-GS-HPV. This direct comparison of substrateconversion to products in assays that differ only in the addition ofLigG or NaGST_(Nu) allows us to conclude that use of the latter enzymehas advantages owing to its greater ability to release HPV from bothGS-HPV epimers under comparable conditions in vitro.

Release of Aromatic Monomers from Lignin Oligomers In Vitro.

The features of this coupled β-etherase assay allowed us to begintesting the ability to remove monomer aromatics from bona fide lignin.Lignin is a heterogeneous, high molecular weight polymer, with onlylimited solubility under the aqueous buffer conditions used for thisassay. Consequently, to increase our chances of observing aromaticproducts under the conditions used for the coupled assay, we usedseveral different lignin oligomers. We also fractionated these materialsto test for release of aromatics from different sized lignin oligomers.This has provided several important new insights into the activity ofβ-etherase enzymes with lignin oligomers and identified opportunitiesfor increasing our understanding of this pathway.

We tested the ability of this enzyme mixture to cleave lignin oligomersthat were derived from an engineered poplar line that contains a highcontent of S units (Stewart et al. 2009). HPS was detected as a productwhen high molecular weight fractions of the HP lignin were used as thesubstrate. This provides direct proof that the enzyme mixture willcleave aromatic oligomers containing S units and that this set ofβ-etherase pathway enzymes are active with lignin oligomers. Given thatthe vast majority of the aromatic units in HP lignin are S units(Stewart et al. 2009), we estimate that the oligomers used in theenzymatic assay had between 40 and 50 aromatic units (Table 9). With theconcentration of lignin oligomer used in this assay (˜2.2 mg mL⁻¹),complete substrate degradation would yield ˜8 mM HPS. The measured HPSconcentration in this assay was 1.0 mM, resulting in a 12.5% yield ofHPS from HP lignin. Thus, it appears that the mixture of enzymes used inthis study, although sufficient for complete cleavage of modeldiaromatic compounds, and of some β-ether links in HP lignin, is notcapable of complete cleavage of all the β-ether linkages in the HPlignin oligomers. It is possible that a heretofore undescribed proteinis required to further process these lignin oligomers, or thatinhibition of enzyme activity was caused by the presence of some of thehigh MW oligomers. Although our findings with the model dimers, andprevious research, indicate that LigD and LigN are sufficient forcomplete oxidation of diaromatic compounds (FIGS. 17A and 17B) (Sato etal. 2009, Hishiyama et al. 2012), it is possible that the seeminglyredundant dehydrogenases LigO and LigL have a higher affinity for higherMW lignin oligomers. Similarly, LigP, a GSH-S-transferase with apparentredundant activity with LigE (Tanamura et al. 2010), may be of interestfor the optimization of in vitro lignin depolymerization.

In the assays using HP lignin as a substrate, we did not detectsyringaresinol as a product, even though this dimer is found in lowabundance in this polymer (Stewart et al. 2009). Existing models for thecomposition of HP lignin predict that syringaresinol is primarilyinternal to the polymer (Stewart et al. 2009). Thus, it is possible thatthe failure to detect syringaresinol as a reaction product reflects theinability of the tested β-etherase enzymes to access and cleave β-etherbonds that are adjacent to a syringaresinol moiety or that the enzymesexhibited only limited exolytic activity, thus never reaching thesyringaresinol unit.

Having established that the coupled enzymatic assay exhibited β-etherasecatalytic activity with high-MW fractions of the HP lignin oligomers, wetested a more complex lignin sample from corn stover as a substrate (MCSlignin). Fractionation of this lignin was also carried out andexperiments with a wider array of lignin fractions were conducted totest for the release of the major aromatic monomers present in thismaterial (G, S and T units). The detection of HPV, HPS, and tricin fromdifferent MCS lignin fractions confirms the observations with theβ-ether linked models that the enzyme set used was active in the releaseof G, S, and T units from lignin. However, tricin was only observed withthe lignin fraction having an average MW of 460 (FIG. 22). Using a crudeassumption that the average aromatic unit in lignin has a MW of 210 andthe known MW of tricin (330), this fraction represents mostly lignindimers or a T unit with at most one or two other S or G unit. Thus, theability of the enzymes to cleave the β-ether linkage next to a flavonoidmoiety appears restricted to lower MW oligomers. In contrast, HPS andHPV were released from MCS lignin in assays using all of the fractionstested (FIGS. 23A and 23B), which we estimate to encompass a range ofoligomers from dimers to 50-unit oligomers (Table 10). The highestmeasured concentration of HPS and HPV corresponded to the ligninfraction with average MW of 1,390, or ˜7 aromatic units (Table 10).Using the same assumption of 210 as the average MW of an aromatic unitin lignin, and the mass of lignin used in the assay (2.2 mg mL⁻¹), weestimate a yield of HPS plus HPV of ˜5%, which is lower than theestimated HPS yield from HP lignin. This lower release of substratesfrom MCS than HP lignin likely reflects the more heterogeneous andcomplex structure of the MCS lignin sample and potential inability ofthe β-etherase pathway enzymes to access and cleave all β-ether bonds inthe polymer.

Taken together, the findings presented here reveal new and excitingfeatures of the β-etherase pathway enzymes. We identified tricin as avaluable flavonoid that can be enzymatically cleaved from β-ether linkedmodels and from low-MW lignin fractions. We also demonstrated β-etheraseactivity with intact lignin oligomers of varying sizes, some of whichmight even be too large to be transported into cells. These findingstherefore provide the first demonstration that in vitro depolymerizationof lignin is possible with β-etherase enzymes, an important step towardsthe development of biotechnological applications designed to derivehigh-value monomeric compounds from bona fide lignin polymers. Theactivity of this set of enzymes on oligomeric substrates provides anopportunity to develop and optimize conditions for aromatic release fromlignin fractions derived from biomass deconstruction chemistries thatare or will be used by industry.

Abbreviations

NAD⁺, nicotinamide adenine dinucleotide; NADH, reduced nicotinamideadenine di-nucleotide; GSH, glutathione; GSSG, glutathione disulfide;GS-HPV, β-S-glutathionyl-γ-hydroxypropiovanillone; GS-HPS, β-S-glutathionyl-γ-hydroxypropiosyringone; HPV, γ-hydroxypropiovanillone;HPS, γ-hydroxypropiosyringone; GGE, guaiacylglycerol-β-guaiacyl ether;GGE-ketone, α-oxidized GGE; GTE, guaiacylglycerol-β-tricin ether;GTE-ketone, α-oxidized GTE; NaGST_(Nu) , Novosphingobium aromaticivoransstrain DSM12444 glutathione lyase; AvGR, Allochromatium vinosum DSM180glutathione reductase; GPC, gel-permeation chromatography.

Example 3

A Heterodimeric β-Etherase Capable of Sterospecifically Breaking theβ-Aryl Ether Bond Commonly Found in Lignin

Summary

This example describes a newly identified enzyme that can cleave themajor β-aryl ether linkage in plant lignin. Lignin is a heterogeneouspolymer of aromatic units that can constitute as much as 30% of aplant's dry cell weight, making it one of the most abundant renewablematerials on Earth. Currently, there are few economical uses for lignin;the polymer is typically disposed of or burned for energy. The aromaticcompounds that make up lignin could potentially be used in the chemical,cosmetic, food, and pharmaceutical industries; however, due largely toits irregular, covalently bonded structure, lignin has historically beendifficult to depolymerize. Consequently, intensive efforts are currentlyaimed at developing chemical, enzymatic, and hybrid methods for derivingsimpler and lower molecular weight products from lignin.

Some sphingomonad bacteria (e.g. Novosphingobium aromaticivorans) canbreak the bonds between aromatic units in the lignin polymer, includingthe β-aryl ether ((3-O-4) bond, the most common linkage between aromaticunits in lignin (typically >50% of the total linkages). The sphingomonadpathway for breaking the β-aryl ether bond involves three initial steps.First, the α-hydroxyl is oxidized by one of several stereospecificNAD⁺-dependent dehydrogenases (LigL, LigN, LigD, LigO). Next,stereospecific β-etherases (LigF, LigE, LigP) replace the β-ether bondof the resulting α-ketone with a thioether bond involving glutathione(GSH), releasing a glutathione conjugated phenylpropanoid. Finally, theglutathione moiety is removed from the GS-phenylpropanoid by either astereospecific (i.e. LigG) or non-stereospecific (i.e. GST_(Nu))glutathione lyase. All of the characterized GSH-dependent β-etherases inthis pathway function as homodimers.

The β-etherases that react with a particular stereoisomer of the β-arylether bond are similar in amino acid sequence to each other. Theseβ-aryl etherases fall into distinct groups that cleave either the R-(LigE and LigP homodimers) or the S-stereoisomers (LigF homodimers), andthe enzymes that cleave the two different stereoisomers of the β-arylether bond are phylogenetically distinct from each other. We report herea heterodimeric β-aryl etherase (BaeA, comprised of the Saro_2872 andSaro_2873 proteins) that cleaves the R-stereoisomer of the β-aryl etherbond (like LigE and LigP), but is composed of polypeptides that are moresimilar in sequence to (but still phylogenetically distinct from) theenzymes in the LigF group.

This expands the known range of enzymes capable of breaking the β-arylether bond commonly found in lignin, some of which may have kinetic orother properties better suited to operating within an in vitro lignindepolymerization system than the previously characterized LigE and LigPenzymes.

Construction of N. aromaticivorans Mutants

Biological Reagents.

All PCR reactions were performed with Herculase II polymerase (AgilentTechnologies, Santa Clara, Calif.). Primers were phosphorylated withpolynucleotide kinase from Promega (Madison, Wis.). All other enzymeswere from New England Biolabs (Ipswich, Mass.). All primers were fromIntegrated DNA Technologies (Coralville, Iowa).

For cloning using the NEBuilder HiFi Assembly system (New EnglandBiolabs), plasmid pK18msB-MCS (Schafer et al. 1994) was amplified usingprimers “pK18msB AseI ampl F” and “pK18msB-MCS XbaI R” to generate thelinear fragment pK18msB-MCS (see Example 1 above).

Strains.

The strains used in the present example are presented in Table 11.

TABLE 1 Novosphingobium aromaticivorans strains used in this example.Strain Genotype Reference 12444Δ1879 DSM 12444 ΔSaro_1879 Examples above12444ΔligE 1244441879 ΔSaro_2405 This example 12444Δ2872 1244441879ΔSaro_2872 This example 12444Δ2873 1244441879 ΔSaro_2873 This example12444ΔligEΔ2872 1244441879 ΔSaro_2405 ΔSaro_2872 This example

TABLE 12 Primers used in genomic modifications and enzyme expression.Name Sequence Notes pK18msB 5′-CTGTCGTGCCAGCTGCATTAATG-3′ (SEQ ID AseIsite AseI ampl F NO: 64) (underlined) native to template pK18msB-5′-GAACAtcTAGAAAGCCAGTCCGCAGAA AC- XbaI site MCS XbaI R 3′ (SEQ ID NO:65) (underlined); lowercase bases do not match template pK18-ligE5′-GTTTCTGCGGACTGGCTTTCTAGATGTTCC Underlined region OvExt FAGTGCTCTACAACCAGTCGTACCACATG-3′ is complementary (SEQ ID NO: 97) topK18msB-MCS pK18-ligE 5′-CGATTCATTAATGCAGCTGGCACGACAGCG Underlinedregion OvExt R AGTTGAACGAAACCTCCTCGTTCATG-3′ (SEQ is complementary IDNO: 98) to pK18msB-MCS Saro2405 5′-GCATCACCGAAGGCATGAAGAAGTAAACG- ligEdel F 3′ (SEQ ID NO: 99) Saro2405 5′-GTGACTCAATTGCCGTCACCCTGAACTTG-3′ligE del R (SEQ ID NO: 100) Saro_28725′-CATCattaATTCGACCTGGCCATAGGACTG-3′ AseI site ampl AseI F2 (SEQ ID NO:101) (underlined); lowercase bases do not match template Saro_28725′-taGttCtaGACCATCTTTTCCGCTGGAGC-3′ XbaI site ampl XbaI R (SEQ ID NO:102) (underlined); lowercase bases do not match template Saro_28725′-GCTTGTCAAGGCCTGGCTTGC-3′ (SEQ ID del R NO: 103) Saro_28725′-TtATCCCTCGATCTCCGCCATGATGAG-3′ lowercase base del F (SEQ ID NO: 104)does not match template Saro_2873- 5′-GTTTCTGCGGACTGGCTTTCTAGATGTTCCCUnderlined region pk18 hifi TACAAGGGAGGGCAGTGAAATGAAGC-3′ (SEQ iscomplementary ampl R ID NO: 105) to pK18msB-MCS Saro_28735′-CATCCCTCGATCTCGTCCATCCGCTGCCCA Underlined region hifi del FTCC-3′ (SEQ ID NO: 106) is complementary to Saro_2873 hifi del RSaro_2873- 5′-CGATTCATTAATGCAGCTGGCACGACAGG Underlined region pk18 hifiGACGAATGATAGACCAGCCACTTCAGG-3′ is complementary ampl F (SEQ ID NO: 107)to pK18msB-MCS Saro_2873 5′-GATGGACGAGATCGAGGGATGAGCGCGCT Underlinedregion hifi del R TCTTTACC-3′ (SEQ ID NO: 108) is complementary toSaro_2873 hifi del F Saro2872 5′-GGCatctgcgaGaccTCCCCAACGGTTGATTTC BsaIsite Ctag BsaI F AG-3′ (SEQ ID NO: 109) (underlined); lowercase bases donot match template Saro2872 5′-CGAGtcATGAGCGCGCTTCTTTACCACG-3′ BspHIsite Ctag BspHI R (SEQ ID NO: 110) (underlined); lowercase bases do notmatch template pVP302K 5′-CTGCGGTCTCGCAGATGGTAAAATTCTG-3′ BsaI site CtagBsaI F (SEQ ID NO: 80) (underlined) pVP302K5′-GGTGATGTCCCATGGTTAATTTCTCCTCTTT NcoI site Ctag NcoI R AATG-3′ (SEQ IDNO: 81) (underlined) Ctag 2872- 5′-CGAGttaTCCCCAACGGTTGATTTCAGG-3′lowercase bases do pVP add (SEQ ID NO: 111) not match template Stop RpVP302K 5′-CATTAAaAGcTTAAACGAATTCGGACTCGG HindIII site Ntag HindIII FTACGC-3′ (SEQ ID NO: 83) (underlined); lowercase bases do not matchtemplate 2872-pVP C 5′-caagcgaaaatctgtattttcagagcgcgatcgcaggaATGAGClowercase bases do to Ntag F GCGCTTCTTTACCACG-3′ (SEQ ID NO: 112) notmatch template pVP302 C to5′-ccaatgcatggtgatggtgatgatggtgatgtcccatGGTTAATT lowercase bases do NtagR TCTCCTCTTTAATG-3′ (SEQ ID NO: 85) not match template Saro28725′-caagcgaaaatctgtattttcagagcgcgatcgcaggaAGCGCG lowercase bases do gNtagR CTTCTTTACCACGG-3′ (SEQ ID NO: 113) not match template Saro28725′-ccaatgcatggtgatggtgatgatggtgatgtaTCATCCCTCG lowercase bases do gNtagF ATCTCCGCCATGATG-3′ (SEQ ID NO: 114) not match template 2872-5′-CTAACTTTGTTATTTTCGGCTTTCTGTTATC Underlined region 3_pVP_HiFi_FCCCAACGGTTGATTTCAGG-3′ (SEQ ID NO: 115) is complementary to pVP302KSaro2872- 5′-GAATTCATTAAAGAGGAGAAATTAACCAT Underlined region3NOTAG_pVP_HiFi_R GGACGAGGTAAGCCTCTATCATTGG-3′ (SEQ ID is complementaryNO: 116) to pVP302K pVP302K- 5′-GGTTAATTTCTCCTCTTTAATGAATTCTGTGHiFi-noTag-R TGAAATTG-3′ (SEQ ID NO: 117) pVP302K-5′-CAGAAAGCCGAAAATAACAAAGTTAGCCT HiFi-ATW-F GAGCTG-3′ (SEQ ID NO: 91)Saro2872- 5′-GTATTTTCAGAGCGCGATCGCAGGAATGG Underlined region3Ntag_pVP_HiFi_R ACGAGGTAAGCCTCTATCATTGG-3′ (SEQ ID is complementary NO:118) to pVP302K pVP302K- 5′-TCCTGCGATCGCGCTCTGAAAATACAGATT HiFi-ATW-RTTCG-3′ (SEQ ID NO: 90) Saro2872- 5′-CGCGgCGCTCACCGTTCTTGC-3′ (SEQ IDLowercase g S14A_R NO: 119) introduces S→A mutation in underlined codonSaro2872- 5′-CCGTTGGGCTCGCCGTGGTAAAGAAG-3′ S14A_F (SEQ ID NO: 120)Saro2873- 5′-GCAAGCCGATGCTCGCGTTGATG-3′ (SEQ ID S15A_R NO: 121)Saro2873- 5′-CAGcGTTGGCATTGGGTTCCCAATGATAG Lowercase c S15A_F AG-3′ (SEQID NO: 122) introduces S→A mutation in underlined codon Saro2873-5′-CAGAGgcGGCATTGGGTTCCCAATGATAG Lowercase gc N14A_F AG-3′ (SEQ ID NO:123) introduces N→A mutation in underlined codon pEU-HiFi-5′-GTGATGATGATGATGATGTCCCATTAAC-3′ ATW-R (SEQ ID NO: 124) pEU-HiFi-5′-TAGTTTAAACGAATTCGAGCTCGG-3′ (SEQ ATW-F ID NO: 125) Saro2872-5′-GGACATCATCATCATCATCACGCATTGGCA Underlined region pEU2394-AGCGAAAATCTGTATTTTCAG-3′ (SEQ ID is complementary HiFi-F NO: 126) to pEUSaro2872- 5′-CCGAGCTCGAATTCGTTTAAACTACGAGTT Underlined region pEU2394-ATCCCCAACGGTTGATTTCAGG-3′ (SEQ ID is complementary HiFi-R NO: 127) topEU pEU-2872- 5′-CATTAACTAACTAGTGTAGTTGTAGAATGT Underlined region fix-RAAAATGTAATGTTGTTGTTGTTTG-3′ (SEQ ID was missing in NO: 128) originallycreated pEU-2872 pEU-2872- 5′-GGACATCATCATCATCATCACGCATTGG-3′ fix-F (SEQID NO: 129) Saro_2873- 5′-CAACTACACTAGTTAGTTAATGGACGAGGT Underlinedregion pEU_HiFi-F AAGCCTCTATCATTGG-3′ (SEQ ID NO: 130) is complementaryto pEU Saro_2873- 5′-CGAGCTCGAATTCGTTTAAACTACTCATCC Underlined regionpEU_HiFi-R CTCGATCTCCGCCATG-3′ (SEQ ID NO: 131) is complementary to pEUpEU2394 F 5′-GTAGTTTAAACGAATTCGAGCTCGGTACC-3′ (SEQ ID NO: 132)

Plasmid for Deleting Saro_2405.

A 3844 bp region of the N. aromaticivorans genome extending from 1501 bpupstream of Saro_2405 to 1503 bp downstream of the gene was amplifiedfrom purified genomic DNA using primers “pK18-ligE OvExt F” and“pK18-ligE OvExt R”, which contain 5′ ends that are complementary to theends of linearized pK18msB-MCS. The genomic DNA fragment was combinedwith linearized pK18msB-MCS using the NEBuilder HiFi Assembly system toproduce plasmid pK18msB-ligE. This plasmid was amplified using kinasephosphorylated primers “Saro2405 ligE del F” and “Saro2405 ligE del R”to produce a linear fragment in which the majority of Saro_2405(including the start codon) was missing. This linear fragment wascircularized using T4 DNA Ligase to generate plasmid pK18msB-ΔligE.

Plasmid for Deleting Saro_2872.

A ˜2813 bp region of the N. aromaticivorans genome extending from 1073bp upstream of Saro_2872 to 954 bp downstream of the gene was amplifiedfrom purified genomic DNA using primers “Saro_2872 ampl AseI F2” and“Saro_2872 ampl XbaI R”, which contain recognition sites for therestriction enzymes AseI or XbaI, respectively, incorporated into their5′ ends. The resulting fragment was digested with AseI and XbaI, thenligated with pK18msB-MCS that had been digested with AseI and XbaI,using T4 DNA Ligase, to form plasmid pK18msB-Saro2872. This plasmid wasamplified using kinase phosphorylated primers “Saro_2872 del R” and“Saro_2872 del F” to produce a linear fragment in which the majority ofSaro_2872 was missing. Since the start codon of Saro_2872 overlaps withthe stop codon of Saro_2873, “Saro_2872 del F” contains a single basemismatch with pK18msB-Saro2872, to inactivate the Saro_2872 start codon,while preserving the Saro_2873 stop codon. This linear fragment wascircularized using T4 DNA Ligase to generate plasmid pK18msB-ΔSaro2872.

Plasmid for Deleting Saro_2873.

˜1100 bp regions from upstream and downstream of Saro_2873 in the N.aromaticivorans genome were separately amplified from purified genomicDNA using primer sets “Saro_2873-pk18 hifi ampl R” and “Saro_2873 hifidel F”, and “Saro_2873-pk18 hifi ampl F” and “Saro_2873 hifi del R”,respectively. These two fragments were combined with linearizedpK18msB-MCS using the NEBuilder HiFi Assembly system to produce plasmidpK18msB-ΔSaro2873, in which the regions that naturally flank Saro_2873in the genome are adjacent to each other.

Deleting Genes from the N. aromaticivorans Genome.

Deletion plasmids were separately mobilized into N. aromaticivorans viaconjugation with Escherichia coli S17-1. For the conjugation, culturesof E. coli S17-1 harboring the plasmid and N. aromaticivorans were grownup overnight in Lysogeny Broth containing kanamycin or GluSis,respectively. Cultures were subcultured and allowed to resumeexponential growth before being harvested by centrifugation. E. coli andN. aromaticivorans cell pellets were washed in lysogeny broth, thenresuspended together into 90 μL lysogeny broth. Conjugations wereallowed to proceed overnight at 30° C. The following day, theconjugations were outgrown in GluSis at 30° C. for >1 h, then platedonto solid GluSis with kanamycin to select for N. aromaticivorans cellsin which the plasmid had incorporated into the genome via homologousrecombination (single crossovers). Single crossovers were confirmedthrough the inability to immediately grow on GluSis containing 10%sucrose.

Single crossovers were cultured in 5 mL of GluSis containing 10% sucroseand shaken at 30° C. until growth commenced (usually after severaldays), which signified loss of the plasmid from the genome via a secondround of homologous recombination. These cultures were streaked ontosolid GluSis+10% sucrose to isolate individual strains that has lost theplasmid (double crossovers), and plasmid loss was confirmed by theinability to grow on GluSis+kanamycin. The absences of the desired geneswere confirmed via PCR performed on isolated genomic DNA and Sangersequencing.

Bacterial Growth Media

E. coli cultures used for cloning were grown in lysogeny broth (LB), andshaken at ˜200 rpm at 37° C. For routine storage and manipulation, N.aromaticivorans cultures were grown in LB or GluSis at 30° C. GluSis isa modification of Sistrom's minimal medium in which the succinate hasbeen replaced by 22.6 mM glucose (see Example 1, above). N.aromaticivorans growth experiments used Standard Mineral Base (SMB)minimal medium, as described in Example 1, except at pH 7.0. Whereneeded to select for plasmids, media were supplemented with 100 μg/mLampicillin, 50 μg/mL kanamycin, or 20 μg/mL chloramphenicol.

N. aromaticivorans Growth Experiments

Starter cultures of N. aromaticivorans were grown in 4 mL SMB containing4 mM vanillate. Experimental cultures were grown in 20-30 mL of SMBcontaining 3 mM vanillate and 1 mM GGE, in 125 mL conical growth flasksshaken at 200 rpm at 30° C. Aliquots (400-600 μL) were removed atspecified time points and filtered through 0.22 μm syringe tip filters(e.g. Whatman Puradisc filters, GE Healthcare) before HPLC analysis ofextracellular aromatics. Every culture was grown at least three times;data shown are from representative cultures.

For the 12444ΔligEΔ2872 and 12444ΔligEΔ2873 cultures, we filtered >2 mLfor the final time points. These samples still contained MPHPV; todetermine which stereoisomer(s) of MPHPV remained present, the sampleswere split into three 400 uL aliquots and combined with 5 mM GSH andeither H₂O, recombinant LigE (90 μg/mL), or recombinant LigF1 (147μg/mL), and incubated at 30° C. for 1 h. These samples were thenanalyzed via HPLC as described below.

Expression and Purification of Recombinant Proteins

Plasmid for Expressing Recombinant Saro_2872.

Saro_2872 was amplified from N. aromaticivorans genomic DNA with theprimers “Saro2872 Ctag BsaI F” and “Saro2872 Ctag BspHI R”. Thisfragment was digested with restriction enzymes BspHI and BsaI. Theexpression vector pVP302K (Gall and Ralph et al. 2014) was amplifiedusing the primers “pVP302K Ctag BsaI F” and “pVP302K Ctag NcoI R”, andthe resulting fragment was digested with BsaI and NcoI. The digestedfragments were ligated using T4 DNA ligase, generating plasmidpVP302K/Ctag-2872, which consists of a T5 promoter followed by thecoding sequences of Saro_2872 (absent the stop codon), the RtxA proteasefrom Vibrio cholerae, and a His₈ tag.

pVP302K/Ctag-2872 was amplified using kinase phosphorylated primers“Ctag 2872-pVP add Stop R” and “pVP302K Ntag HindIII F”. This fragmentwas circularized using T4 DNA ligase to generate plasmidpVP302K/Untagged2872, in which a stop codon has been introduced directlyafter Saro_2872.

pVP302K/Untagged2872 was amplified via PCR using kinase phosphorylatedprimers “2872-pVP C to Ntag F” and “pVP302 C to Ntag R”. The amplifiedfragment was circularized using T4 DNA ligase to generate plasmidpVP302K/Ntag-2872, which contains a T5 promoter followed by codingsequences for a His₈-tag, a tobacco etch virus (Tev) proteaserecognition site and Saro_2872.

Plasmids for Expressing Recombinant Saro_2872 and Saro_2873 Together(BaeA).

To Express BaeA Containing a His₈-Tag on the N-Terminus of Saro_2872:

We first generated a strain of N. aromaticivorans in which a codingsequence for a His₈-tag was incorporated into the genome so thatcellular copies of Saro_2872 protein would contain a His₈-tag on theirN-terminus. pK18msB-Saro2872 was amplified via PCR using kinasephosphorylated primers “Saro2872 gNtag R” and “Saro2872 gNtag F”, togenerate a fragment containing Saro_2873 (with its stop codon), followedby a coding sequence for a His₈-tag, then a Tev protease recognitionsite, then Saro_2872 (missing its native start codon). This fragment wascircularized using T4 DNA ligase to generate plasmid pK18msB-H₈Saro2872.pK18msB-H₈Saro2872 was mobilized into strain 1244442872 via conjugationfrom E. coli S17-1, and a strain of N. aromaticivorans (12444-H₈2872)containing the coding sequence for Saro_2872 containing an N-terminalHis₈-tag was generated and isolated using homologous recombination asdescribed above for generating deletion mutants.

We ran PCR using genomic DNA from strain 12444-H₈2872 as template withprimers “2872-3_pVP_HiFi_F” and “Saro2872-3NOTAG_pVP_HiFi_R” to generatea fragment containing the coding sequence for Saro_2873 (with stop codonintact), followed by the coding sequence for a His₈-tag, then forSaro_2872 (missing its start codon), with extensions on the ends of thefragment that are complementary to plasmid pVP302K. pVP302K wasamplified via PCR using the primers “pVP302K-HiFi-noTag-R” and“pVP302K-HiFi-ATW-F”. These two fragments were combined using theNEBuilder HiFi Assembly system to create plasmid pVP302K/2873-H2872.

To Express BaeA Containing a His₈-Tag on the N-Terminus of Saro_2873:

We ran PCR using genomic DNA from strain 12444Δ1879 as template withprimers “2872-3_pVP_HiFi_F” and “Saro2872-3Ntag_pVP_HiFi_R” to generatea fragment containing the native genomic organization of the Saro_2873and Saro_2872 genes, with extensions on the ends of the fragment thatare complementary to plasmid pVP302K. pVP302K was amplified via PCRusing the primers “pVP302K-HiFi-ATW-R” and “pVP302K-HiFi-ATW-F”. Thesetwo fragments were combined using the NEBuilder HiFi Assembly system tocreate plasmid pVP302K/H2873-2872.

To Express BaeA Mutants:

To generate mutant 2:S14A, pVP302K/2873-H2872 was amplified by kinasephosphorylated primers “Saro2872-S14A_R” and “Saro2872-S14A_F”. Togenerate mutant 3:S15A, pVP302K/H2873-2872 was amplified by kinasephosphorylated primers “Saro2873-S15A_R” and “Saro2873-S15A_F”. Togenerate mutant 3:N14A, pVP302K/2873-H2872 was amplified by kinasephosphorylated primers “Saro2873-S15A_R” and “Saro2873-N14A_F”. Theselinear fragments were separately circularized using T4 DNA ligase togenerate plasmids pVP302K/2873-H2872(S14A), pVP302K/H2873(S15A)-2872,and pVP302K/2873(N14A)-H2872, respectively.

To generate mutant 2:14A/3:S15A, pVP302K/2873-H2872(S14A) was amplifiedby kinase phosphorylated primers “Saro2873-S15A_R” and“Saro2873-S15A_F”. The linear fragment was circularized using T4 DNAligase to generate plasmid pVP302K/2873(S15A)-H2872(S14A).

Plasmids for Expressing Recombinant Saro_2873.

We amplified plasmids pVP302K/2873-H2872 and pVP302K/H2873-2872 via PCRusing kinase phosphorylated primers “pVP302K-HiFi-ATW-F” and “Saro_2872del F”. These linear fragments were separately circularized using T4 DNAligase to generate plasmids pVP302K/Untagged2873 and pVP302K/Ntag-2873,respectively.

Expression and Purification of Recombinant Enzymes.

Recombinant proteins were expressed using the plasmids described abovein E. coli B834 containing plasmid pRARE2 (Novagen) grown for ˜25 hoursat 25° C. in ZYM-5052 Autoinduction Medium containing kanamycin andchloramphenicol. Recombinant proteins were purified using a Ni²⁺-NTAcolumn as described in Example 1 above, except using gravity-flowcolumns instead of an FPLC system. After removal of His₈-tags using Tevprotease, recombinant proteins retained a Ser-Ala-Ile-Ala-Gly-peptide ontheir N-termini, derived from the linker between the protein and the Tevprotease recognition site. Recombinant LigF1 was purified as previouslydescribed (Gall and Ralph et al. 2014).

Recombinant enzyme concentrations were determined via the Bradfordmethod (absorbance at 595 nm), using known concentrations of bovineserum albumin as standards (Thermo Scientific) and protein assay dyereagent from Biorad.

Cell-Free Synthesis of Saro_2872 and Saro_2873

Plasmid for Expressing Saro_2872 in a Cell-Free System.

Plasmid pEU-NGFP (Goren et al. 2009) was amplified via PCR using primers“pEU-HiFi-ATW-R” and “pEU-HiFi-ATW-F”to generate a linear fragment inwhich the gene for Green Fluorescent Protein has been removed.pVP302K/Ntag-2872 was amplified via PCR using primers“Saro2872-pEU2394-HiFi-F” and “Saro2872-pEU2394-HiFi-R” to generate alinear fragment containing the coding sequence for the Tev proteaserecognition site followed by Saro_2872. These linear fragments werecombined using the NEBuilder HiFi Assembly system to create a plasmidthat was missing a short sequence upstream of the translational startsite. To add this sequence, we amplified the plasmid using kinasephosphorylated primers “pEU-2872-fix-R” and “pEU-2872-fix-F”. The linearfragment was circularized using T4 DNA ligase to form plasmid pEU-H2872,which contains a sequence for a His₆-tag, followed by a Tev proteaserecognition site, then Saro_2872.

Plasmid for Expressing Saro_2873 in a Cell-Free System.

N. aromaticivorans genomic DNA was amplified via PCR using primers“Saro_2873-pEU_HiFi-F” and “Saro_2873-pEU_HiFi-R” to generate a linearfragment containing Saro_2873 with ends that are complementary to pEU.pEU-H2872 was amplified via PCR using primers “pEU-2872-fix-R” and“pEU2394 F” to generate a linear fragment in which the sequences for theHis₆-tag, the Tev protease recognition site, and Saro_2872 were removed.These linear fragments were combined using the NEBuilder HiFi Assemblysystem to create plasmid pEU-2873.

Cell-Free Protein Synthesis.

Cell-free protein synthesis was run essentially as previously described(Makino et al. 2014). The Saro_2872 polypeptide contained a His₆-tag anda Tev protease recognition site on its N-terminus that were not removed.The Saro_2873 polypeptide was synthesized in its native form.Synthesized polypeptides were not purified from the synthesis reactionmixture; assays for enzymatic activity were performed by adding aliquotsdirectly from the synthesis reaction. Concentrations of the Saro_2872and Saro_2873 polypeptides in the reaction mixtures were approximatedusing the intensities of the bands in an SDS-PAGE gel.

Assays to Determine Activities and Stereospecificties of Saro_2872 andSaro_2873

0.1 mM racemic (β(S) and β(R)) MPHPV was combined with 5.8 mMglutathione (GSH) in reaction buffer (RB; 25 mM Tris-HCl (pH 8.0) and 25mM NaCl). Cell-free protein synthesis mixtures containing Saro_2872 andSaro_2873 were added individually or together to the MPHPV/GSH solutionsto achieve concentrations of ˜24 nM of each polypeptide. These 625 μLreactions were incubated at 30° C. for 24 h to several days. Each wasthen split into 190 μL aliquots and combined with an additional 2.3 mMGSH and either H₂O, 151 μg/mL LigE (Saro_2405), or 184 μg/mL LigF1(Saro_2091). These 212 μL reactions were incubated at 30° C. for severalhours, then analyzed via HPLC.

Kinetics of the Enzymatic Cleavage of β(R)-MPHPV.

Various concentrations of racemic MPHPV (equal amounts of the β(S)- andβ(R)-stereoisomers) were combined with 5 mM GSH in RB. At time zero, 100μL of a given enzyme in RB+5 mM GSH was combined with 1000 μL of theracemic MPHPV/GSH sample at 25° C. (both samples were equilibrated to25° C. before mixing). Final concentrations of β(R)-MPHPV in eachreaction were 0.0045, 0.010, 0.017, 0.068, or 0.13 mM. Final enzymeconcentrations were 18 nM BaeA, 23 nM BaeA (2:S14A), 22 nM BaeA(3:S15A), 24 nM BaeA (2:S14A/3:S15A), 98 nM BaeA (3N14A), or 70 nM LigE(Saro_2405) (all concentrations are for the dimeric enzyme, except forLigE, which is the concentration of the monomer). At specified timepoints, 200 μL of a reaction was removed and combined with 40 μL of 1 MHCl (Acros Organics) to stop the reaction before HPLC analysis toquantify GS-HPV formed. Control experiments were allowed to proceed forseveral hours to ensure that only the β(R)-MPHPV in the reactionmixtures was being reacted with in these experiments.

HPLC Analysis.

Analysis and quantification of aromatic compounds were performed usingan Ultra AQ C18 5 μm column (Restek) attached to a System Gold HPLC(Beckman Coulter) with running buffers and methods described inExample 1. The eluent was analyzed for light absorbance between 191 and600 nm, and absorbances at 280 nm were used for quantification ofaromatic metabolites by comparing peak areas to those of standards.

Results

An N. aromaticivorans Saro_2405 (ligE) Deletion Mutant can CompletelyMetabolize Erythro-GGE.

LigE from N. aromaticivorans is capable of stereospecifically breakingthe β-aryl ether bond of the β(R) stereoisomers of MPHPV and otherdi-aromatic compounds in vitro. To investigate the in vivo role of LigEin N. aromaticivorans, we constructed a strain in which the gene forLigE (Saro_2405) was deleted from the genome (12444ΔligE), and grew it,along with its parent strain (12444Δ1879), in a medium containingvanillate and erythro-GGE.

As expected from the examples provided above, 12444Δ1879 completelyconsumed both the vanillate and the GGE. Metabolism of GGE proceededthrough several intermediates, including both β(R) and β(S) MPHPV, whichtransiently appeared in the medium, then were taken back up by the cells(FIG. 24, panel A). Consistent with our examples above, only a traceamount of guaiacol appeared in the extracellular medium, and theglutathione conjugate GS-HPV was never observed in the medium.

As the gene product of Saro_2405 is the only predicted homologue of LigEand LigP in N. aromaticivorans, and LigE and LigP are the onlysphingomonad enzymes known to be capable of breaking the β(R)stereoisomer of the β-aryl ether bond, we expected that strain12444ΔligE would be incapable of fully metabolizing erythro-GGE.However, although MPHPV consistently disappeared from the medium slowerfor 12444ΔligE than for 12444Δ1879, 12444ΔligE was capable of completelyremoving racemic MPHPV from the medium (FIG. 24, panel B). As MPHPVdisappeared from the medium, HPV accumulated in the medium up to aconcentration roughly equal to the initial erythro-GGE concentration,suggesting that essentially all of the GGE was metabolized through MPHPVand into HPV. These results suggest that 12444ΔligE contains an enzymecapable of breaking the β-aryl ether bond of β(R)-MPHPV.

Saro_2872 and Saro_2873 are Required for Cleavage of β(R)-MPHPV in N.aromaticivorans 12444ΔligE.

As LigE, LigP, and LigF are all classified as glutathioneS-transferases, we expected that a glutathione S-transferase wasreacting with β(R)-MPHPV in the 12444ΔligE strain. We thus investigatedSaro_2872 and Saro_2873, which are annotated as coding forglutathione-S-transferases and are located in a gene cluster withSaro_2865, which codes for one of the two LigF isoforms in N.aromaticivorans. We separately deleted Saro_2872 and Saro_2873 from thegenome of 12444ΔligE and found that neither of the resulting strains,12444ΔligEΔ2872 and 12444ΔligEΔ2873, respectively, could fullymetabolize erythro-GGE (FIG. 24, panels D and E). Each strainaccumulated MPHPV in its medium to a concentration roughly one-half ofthe medium's initial erythro-GGE concentration, suggesting that theywere deficient in metabolizing MPHPV.

Our method of analysis does not distinguish between the β(R) and β(S)stereoisomers of MPHPV. Therefore, to determine which stereoisomer(s) ofMPHPV remained unreacted in the media of the 12444ΔligEΔ2872 and12444ΔligEΔ2873 cultures, the spent media were filtered, and individualaliquots were combined with H₂O, or recombinant LigF or LigE, which areknown to react stereospecifically with the β(S) or β(R) isomers ofMPHPV, respectively (FIG. 25, panels F-K). Addition of LigF to the spentmedia samples resulted in the conversion of a small amount (<10%) ofMPHPV into GS-HPV and guaiacol (FIG. 25, panels G,J), suggesting thatsome of the unconsumed MPHPV was the β(S) isomer. However, addition ofLigE resulted in conversion of most of the MPHPV into GS-HPV andguaiacol (FIG. 25, panels H,K), suggesting that a large fraction of theMPHPV was the β(R) isomer. These results suggest that 12444ΔligErequires both Saro_2872 and Saro_2873 for complete metabolism of MPHPV,particularly the β(R) isomer.

To determine whether both Saro_2872 and Saro_2873 are necessary forcomplete metabolism of MPHPV in an N. aromaticivorans strain with afunctional LigE, we deleted Saro_2872 from 12444Δ1879. The resultingstrain (12444Δ2872) was capable of fully metabolizing erythro-GGE (FIG.24, panel C), though it removed the MPHPV from the medium slower than12444Δ1879 (FIG. 24, panel A), similar to 12444ΔligE (FIG. 24, panel B).Thus, it appears that either LigE or a combination of Saro_2872 andSaro_2873 is sufficient for completely metabolizing β(R)-MPHPV.

The Saro_2872 and Saro_2873 Polypeptides Form a Heterodimer that isStereospecific for β(R)-MPHPV.

As our genetic results suggested that the Saro_2872 and Saro_2873 geneproducts contribute to cleavage of β(R)-MPHPV in N. aromaticivorans, wesought to express these proteins and test them for this activity invitro.

Initial attempts to individually express and purify the Saro_2872 andSaro_2873 polypeptides recombinantly in Escherichia coli wereunsuccessful. We thus separately expressed each polypeptide using acell-free protein synthesis system. When the polypeptides wereindividually combined with racemic (β(R) and β(S)) MPHPV, a trace amountof GS-HPV appeared in the reactions (<1% of the initial MPHPVconcentration), but essentially all of the MPHPV remained unreacted(FIG. 26, panels A-C), even after several days.

The lack of activity of the Saro_2872 and Saro_2873 polypeptides, andour observation that the Saro_2872 and Saro_2873 ORFs overlap in the N.aromaticivorans genome, led us to hypothesize that the polypeptides mayform a heterodimer. Indeed, when we combined the separately preparedSaro_2872 and Saro_2873 polypeptides with each other and with racemicMPHPV, half of the MPHPV was converted into GS-HPV and guaiacol (FIG.26, panel D). To determine which stereoisomer(s) of MPHPV remainedunreacted in this reaction, we split the reaction mixture and addedrecombinant LigE (Saro_2405) or LigF1 (Saro_2091). Upon addition ofLigE, no change in the amounts of MPHPV, GS-HPV, or guaiacol wasobserved (FIG. 26, panel E). Upon addition of LigF1, the remaining MPHPVin the reaction mixture was converted into GS-HPV and guaiacol (FIG. 26,panel F), suggesting that the MPHPV remaining after the reaction ofracemic MPHPV with the mixture of Saro_2872 and Saro_2873 was the β(S)stereoisomer.

To generate larger amounts of the Saro_2872-Saro_2873 complex than waspossible with the cell-free system, we attempted to express theSaro_2872 and Saro_2873 polypeptides together from a single expressionvector in E. coli. Despite the fact that only one of the polypeptidescontained a His₈-tag on its N-terminus, two polypeptides from the E.coli cell lysate, corresponding to the expected sizes of Saro_2872 andSaro_2873, reversibly bound to a Ni²⁺-NTA column, consistent withSaro_2872 and Saro_2873 forming a heterodimer. Indeed, the purifiedrecombinant protein ran as a single peak that corresponded to a dimer ingel permeation chromatography experiments (FIG. 27).

In reactions similar to those performed with the cell-free generatedpolypeptides, we found that the recombinantly generatedSaro_2872-Saro_2873 complex reacted specifically with β(R)-MPHPV, anddid not react with β(S)-MPHPV. Because the Saro_2872-Saro_2873heterodimer is a β-aryl etherase, we call the heterodimer BaeA. The factthat BaeA has the same stereospecificity as LigE (for β(R)-MPHPV) iscurious, as both Saro_2872 and Saro_2873 cluster much closer to thepreviously characterized LigF enzymes than to the previouslycharacterized LigE enzymes in a phylogenetic analysis (FIG. 28). Thisfinding likely has implications for the evolution of the enzymaticability to break the two stereoisomers of the β-aryl ether bond oflignin.

The Saro_2873 Subunit is Much More Catalytically Active than theSaro_2872 Subunit in BaeA.

We sought to gain insight into the relative activities of the Saro_2872and Saro_2873 subunits in BaeA by independently inactivating one or theother of the subunits. Previous work found that LigF from Sphingobiumsp. SYK-6 (SLG_08650) contains a serine residue in its active site(Ser¹⁴) that is important for reacting withβ(S)-(1′-formyl-3′-methoxyphenoxy)-γ-hydroxypropioveratrone (an analogueof MPHPV): mutation of the serine had a dramatic effect on the reactionrate, although it was unclear whether the effect was from changes insubstrate binding, turnover, or both (Helmich et al. 2016). This serineresidue is conserved in all the previously characterized LigF enzymes,and in both Saro_2872 (Ser¹⁴) and Saro_2873 (Ser¹⁵) (FIG. 29). Wemutated these serines into alanines separately (2:S14A and 3:S15A) andtogether (2:S14A/3:S15A) in BaeA and assayed the variant enzymes invitro, along with wild-type BaeA. As we did not know the relativeactivities of the two subunits in BaeA, we initially calculated kineticparameters for the enzymes using the concentrations of the dimers (andnot the total concentrations of the individual putative active sites).

Wild-type BaeA and the three serine mutants all had the same k_(cat)value, suggesting that these serine residues are not involved inenzymatic turnover (Table 13). However, the variants in which Ser¹⁵ inSaro_2873 was mutated (3:S15A and 2:S14A/3:S15A) had K_(M) values thatwere 5 to 6-fold higher than that of the wild-type enzyme, suggestingthat these variants bound the substrate weaker than the wild-typeenzyme. The 2:S14A variant had the same K_(M) value as the wild-typeenzyme, which, along with the lack of an effect on k_(cat), implies thatSer¹⁴ in Saro_2872 is not involved in catalysis by BaeA.

TABLE 13 Kinetic parameters for the enzymatic conversion of β(R)-MPHPVinto GS-HPV k_(cat) K_(M) kc_(at)/K_(M) Protein (s⁻¹) (μM) (mM⁻¹s⁻¹)BaeA 2.9 ± 0.3 20 ± 3 150 ± 30  BaeA (2:S14A) 2.8 ± 0.2 22 ± 3 130 ± 20 BaeA (3:S15A) 2.4 ± 0.3 100 ± 20 23 ± 6  BaeA 2.8 ± 0.5 120 ± 30 23 ± 8 (2:S14A/3:S15A) BaeA (3:N14A) 0.14 ± 0.02 250 ± 60 0.5 ± 0.2 LigE 0.68 ±0.06  7 ± 1 100 ± 20 

Analysis of the structure of LigF from Sphingobium sp. SYK-6 (PDB 4xt0)shows that the side-chain amide nitrogen of Asn¹³ is withinhydrogen-bonding distance (3.3 Å) of the bound glutathione thiol group;an analogous asparagine is present in all of the previouslycharacterized LigF enzymes, and in Saro_2873 (Asn¹⁴) (FIG. 29).(Saro_2872 has an Ala in this position (FIG. 29).) Active siteasparagine residues are known or predicted to be involved in catalysisin other glutathione S-transferases. We mutated Asn¹⁴ in Saro_2873 intoAla and found that the resulting BaeA variant (3:N14A) had a k_(cat)value ˜20-fold lower and a K_(M) value ˜12.5-fold higher than wild-typeBaeA (Table 13), suggesting that this residue is critical in bothsubstrate binding and turnover in BaeA. We assume that mutation of thisresidue only affects the active site of the Saro_2873 subunit and doesnot have any long range effects on the active site of Saro_2872 or theoverall folding or structure of the dimer; indeed, all mutant versionsof BaeA used in this study ran as dimers in gel permeationchromatography (FIG. 27), similar to the wild-type enzyme, suggestingthat the mutations did not affect the overall folding of the proteins orthe binding between subunits.

The fact that mutation of a single residue in the Saro_2873 subunit hadsuch a dramatic effect on the overall catalysis of BaeA suggests thatSaro_2873 is the catalytically dominant subunit of the dimer, and that,if the Saro_2872 subunit has any activity in BaeA, it is <˜5% of theactivity of the Saro_2873 subunit.

Catalytic Comparison of BaeA and LigE.

To directly compare catalysis between BaeA and LigE (Saro_2405), we alsoanalyzed recombinant LigE in our in vitro reaction system. We found thatLigE had a ˜4-fold lower k_(cat) value and a ˜3-fold lower K_(M) valuethan BaeA, leading to a catalytic efficiency (k_(cat)/K_(M)) for LigEthat is slightly lower than that of BaeA (Table 13).

REFERENCES

-   Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I.    W., Echols, N., Headd, J. J., Hung, L.-W., Kapral, G. J.,    Grosse-Kunstleve, R. W., et al. (2010). PHENIX: a comprehensive    Python-based system for macromolecular structure solution. Acta    Crystallogr. D Biol. Crystallogr. 66, 213-221.-   Adler E (1977) Lignin chemistry—past, present and future. Wood Sci    Technol 11(3):169-218.-   Adler E: Structural elements of lignin. Industrial & Engineering    Chemistry 1957, 49:1377-1383.-   Adler E, Eriksoo E: Guaiacylglycerol and its β-guaiacyl ether. Acta    chemica Scandinavica 1955, 9:341-342.-   Afonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J.,    Moriarty, N. W., Mustyakimov, M., Terwilliger, T. C., Urzhumtsev,    A., Zwart, P. H., and Adams, P. D. (2012). Towards automated    crystallographic structure refinement with phenix sefine. Acta    Crystallogr. D Biol. Crystallogr. 68, 352-367.-   Akiyama T, Sugimoto T, Matsumoto Y, Meshitsuka G: Erythro/threo    ratio of β-O-4 structures as an important structural characteristic    of lignin. I: Improvement of ozonation method for the quantitative    analysis of lignin side-chain structure. Journal of Wood Science    2002, 48:210-215.-   Bubeck P, Winkler M, Bautsch W (1993) Rapid cloning by homologous    recombination in vivo. Nucleic Acids Res 21(15):3601-3602.-   Bunkóczi, G., and Read, R. J. (2011). Improvement of    molecular-replacement models with Sculptor. Acta Crystallogr. D    Biol. Crystallogr. 67, 303-312.-   Bryksin A V, Matsumura I: Overlap extension PCR cloning: a simple    and reliable way to create recombinant plasmids. Biotechniques 2010,    48:463-465.-   Casanas, A., Warshamanage, R., Finke, A. D., Panepucci, E., Olieric,    V., Nöll, A., Tampé, R., Brandstetter, S., Forster, A., Mueller, M.,    et al. (2016). EIGER detector: application in macromolecular    crystallography. Acta Crystallogr D Struct Biol 72, 1036-1048.-   Cohen-Bazire G, Sistrom W R, Stanier R Y (1957) Kinetic studies of    pigment synthesis by non-sulfur purple bacteria. J Cell Comp Physiol    49(1):25-68.-   Crawford R L, Kirk T K, Harkin J M, McCoy E (1973) Bacterial    cleavage of an arylglycerol-β-aryl ether bond. Appl Microbiol    25(2):322-324.-   del Rio J C, Rencoret J, Prinsen P, Martinez A T, Ralph J, Gutierrez    A: Structural characterization of wheat straw lignin as revealed by    analytical pyrolysis, 2D-NMR, and reductive cleavage method. Journal    of Agricultural and Food Chemistry 2012, 60:5922-5935.-   Doherty A J, Ashford S R, Brannigan J A, Wigley D B (1995) A    superior host strain for the over-expression of cloned genes using    the T7 promoter based vectors. Nucleic Acids Res 23(11):2074-2075.-   Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for    molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60,    2126-2132.-   Fredrickson J K, Brockman F J, Workman D J, Li S W, Stevens T    O (1991) Isolation and characterization of a subsurface bacterium    capable of growth on toluene, naphthalene, and other aromatic    compounds. Appl Environ Microbiol 57(3):796-803.-   Fredrickson J K, et al. (1995) Aromatic-degrading Sphingomonas    isolates from the deep subsurface. Appl Environ Microbiol    61(5):1917-1922.-   Gall D L, Kim H, Lu F, Donohue T J, Noguera D R, Ralph J:    Stereochemical features of glutathione-dependent enzymes in the    Sphingobium sp. strain SYK-6 β-aryl etherase pathway. J Biol Chem    2014, 289:8656-8667.-   Gall D L, Ralph J, Donohue T J, Noguera D R: A group of    sequence-related sphingomonad enzymes catalyzes cleavage of β-aryl    ether linkages in lignin β-guaiacyl and β-syringyl ether dimers.    Environmental Science & Technology 2014, 48:12454-12463.-   Gall D L, Ralph J, Donohue T J, Noguera D R: Biochemical    transformation of lignin for deriving valued commodities from    lignocellulose. (In Review). Current Opinion in Biotechnology 2017.-   Gay P, Le Coq D, Steinmetz M, Berkelman T, Kado C I: Positive    selection procedure for entrapment of insertion sequence elements in    gram-negative bacteria. J Bacteriol 1985, 164(2):918-921.-   Goren M A, Nozawa A, Makino S, Wrobel R, Fox B G: Cell-free    translation of integral membrane proteins into unilamelar liposomes.    Meth. Enzymol. 2009, 463:647-673.-   Grabber J H, Ralph J, Hatfield R D, Quideau S, Kuster T, Pell A N.    Dehydrogenation polymer-cell wall complexes as a model for lignified    grass walls. J. Agric. Food Chem., 1996, 44(6):1453-1459.-   Helmich K E, Pereira J H, Gall D L, Heins R A, McAndrew R P, Bingman    C, Deng K, Holland K C, Noguera D R, Simmons B A, et al.: Structural    basis of stereospecificity in the bacterial enzymatic cleavage of    β-aryl ether bonds in lignin. Journal of Biological Chemistry 2016,    291:5234-5246.-   Higuchi T: Lignin structure and morphological distribution in plant    cell walls. In Lignin biodegradation: microbiology, chemistry and    potential applications. Edited by Kirk T K, Higuchi T, Chang H: CRC    Press; 1980:1-20. vol I.-   Hishiyama S, Otsuka Y, Nakamura M, Ohara S, Kajita S, Masai E,    Katayama Y: Convenient synthesis of chiral lignin model compounds    via optical resolution: four stereoisomers of    guaiacylglycerol-β-guaiacyl ether and both enantiomers of    3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxy-phenoxy)-propan-1-one    (erone). Tetrahedron Letters 2012, 53:842-845.-   Horton R M: In vitro recombination and mutagenesis of DNA: SOEing    together tailor-made genes. Methods in molecular biology (Clifton,    N.J.) 1993, 15:251-261.-   Horton R M, Cai Z, Ho S N, Pease L R: Gene splicing by overlap    extension: tailor-made genes using the polymerase chain reaction.    Biotechniques 2013, 54:129-133.-   Kabsch, W. (2010). XDS. Acta Crystallogr. D Biol. Crystallogr. 66,    125-132.-   Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: a novel method for rapid    multiple sequence alignment based on fast Fourier transform. Nucleic    Acids Res. 2002, 9(14):3059-3066.-   Kontur W S, Bingman C A, Olmsted C N, Wassarman D R, Ulbrich A, Gall    D L, Smith R W, Yusko L M, Fox B G, Noguera D R, Coon J J, Donohue T    J: Novosphingobium aromaticivorans uses a Nu-class glutathione    S-transferase as a glutathione lyase in breaking the β-aryl ether    bond of lignin. J. Biol. Chem. 2018, 293: 4955-4968.-   Lan W, Lu F C, Morreel K, Rencoret J, Del Rio J C, Zakai U, Jones D,    Zhu Y M, Boerjan W, Ralph J: Tricin: A novel monomer in grass    lignins. Abstracts of Papers of the American Chemical Society 2014,    247.-   Lan W, Lu F C, Regner M, Zhu Y M, Rencoret J, Ralph S A, Zakai U I,    Morreel K, Boerjan W, Ralph J: Tricin, a flavonoid monomer in    monocot lignification. Plant Physiology 2015, 167:1284-U1265.-   Lan W, Morreel K, Lu F C, Rencoret J, del Rio J C, Voorend W,    Vermerris W, Boerjan W, Ralph J: Maize tricin-oligolignol    metabolites and their implications for monocot lignification. Plant    Physiology 2016, 171:810-820.-   Larkin M A, Blackshields G, Brown N P, Chenna R, McGettigan P A,    McWilliam H, Valentin F, Wallace I M, Wilm A, Lopez R, Thompson J D,    Gibson T J, Higgins D G. (2007). Clustal W and Clustal X version    2.0. Bioinformatics, 23, 2947-2948.-   Lewis N G, Yamamoto E: Lignin—occurrence, biogenesis and    biodegradation. Annual Review of Plant Physiology and Plant    Molecular Biology 1990, 41:455-496.-   Makino S, Beebe E T. Markley J L, Fox B G: Cell-free protein    synthesis for functional and structural studies. Methods Mol. Biol.    2014, 1091:161-178.-   Masai E, Katayama Y, Nishikawa S, Yamasaki M, Morohoshi N, Haraguchi    T: Detection and localization of a new enzyme catalyzing the β-aryl    ether cleavage in the soil bacterium (Pseudomonas paucimobilis    SYK-6). Febs Letters 1989, 249:348-352.-   Masai E, Kubota S, Katayama Y, Kawai S, Yamasaki M, Morohoshi N:    Characterization of the Ca-dehydrogenase gene involved in the    cleavage of β-aryl ether by Pseudomonas paucimobilis. Bioscience    Biotechnology and Biochemistry 1993, 57:1655-1659.-   Masai E, Katayama Y, Kubota S, Kawai S, Yamasaki M, Morohoshi N: A    bacterial enzyme degrading the model lignin compound β-etherase is a    member of the glutathione-S-transferase superfamily. Febs Letters    1993, 323:135-140.-   Masai E, Ichimura A, Sato Y, Miyauchi K, Katayama Y, Fukuda M: Roles    of the enantioselective glutathione S-transferases in cleavage of    β-aryl ether. Journal of Bacteriology 2003, 185:1768-1775.-   Masai E, Katayama Y, Fukuda M (2007) Genetic and biochemical    investigations on bacterial catabolic pathways for lignin-derived    aromatic compounds. Biosci Biotechnol Biochem 71(1):1-15.-   Mashiyama, S. T., Malabanan, M. M., Akiva, E., Bhosle, R.,    Branch, M. C., Hillerich, B., Jagessar, K., Kim, J., Patskovsky, Y.,    Seidel, R. D., et al. (2014). Large-scale determination of sequence,    structure, and function relationships in cytosolic glutathione    transferases across the biosphere. PLoS Biol. 12, e1001843.-   McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D.,    Storoni, L. C., and Read, R. J. (2007). Phaser crystallographic    software. J Appl Crystallogr 40, 658-674.-   Moore D D: Current protocols in molecular biology. Edited by Ausubel    F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A,    Struhl K: John Wiley & Sons; 2003.-   Notomista E, et al. (2011) The marine isolate Novosphingobium sp.    PP1Y shows specific adaptation to use the aromatic fraction of fuels    as the sole carbon and energy source. Microb Ecol 61(3):582-594.-   Ohta Y, Nishi S, Hasegawa R, Hatada Y (2015) Combination of six    enzymes of a marine Novosphingobium converts the stereoisomers of    β-O-4 lignin model dimers into the respective monomers. Sci Rep    5:15105.-   Palamuru S, et al. (2015) Phylogenetic and kinetic characterization    of a suite of dehydrogenases from a newly isolated bacterium, strain    SG61-1L, that catalyze the turnover of guaiacylglycerol-β-guaiacyl    ether stereoisomers. Appl Environ Microbiol 81(23):8164-8176.-   Pal, R., Bhasin, V. K., and Lal, R. (2006). Proposal to reclassify    [Sphingomonas] xenophaga Stolz et al. 2000 and [Sphingomonas]    taejonensis Lee et al. 2001 as Sphingobium xenophagum comb. nov. and    Sphingopyxis taejonensis comb. nov., respectively. Int. J. Syst.    Evol. Microbiol. 56, 667-670.-   Patskovsky Y, et al. PDB ID: 4mzw Crystal structure of nu-class    glutathione transferase Yghu from Streptococcus sanguinis SK36,    complex with glutathione disulfide, target EFI-507286.    doi:10.2210/pdb4mzw/pdb.-   Pereira J H, Heins R A, Gall D L, McAndrew R P, Deng K, Holland K C,    Donohue T J, Noguera D R, Simmons B A, Sale K L, et al.: Structural    and biochemical characterization of the early and late enzymes in    the lignin β-aryl ether cleavage pathway from Sphingobium sp. SYK-6.    Journal of Biological Chemistry 2016, 291:10228-10238.-   Pettersen E F, et al. (2004) UCSF Chimera—a visualization system for    exploratory research and analysis. J Comput Chem 25(13):1605-1612.-   The PyMOL Molecular Graphics System, Version 1.8.2.1 Schrödinger,    LLC Available at: https://www.pymol.org/.-   Rahimi A, Azarpira A, Kim H, Ralph J, Stahl S S: Chemoselective    metal-free aerobic alcohol oxidation in lignin. Journal of the    American Chemical Society 2013, 135:6415-6418.-   Rahimi A, Ulbrich A, Coon J J, Stahl S S: Formic-acid-induced    depolymerization of oxidized lignin to aromatics. Nature 2014,    515:249-252.-   Ralph J, Peng J P, Lu F C, Hatfield R D, Helm R F: Are lignins    optically active? Journal of Agricultural and Food Chemistry 1999,    47:2991-2996.-   Reiter J, Strittmatter H, Wiemann L O, Schieder D, Sieber V:    Enzymatic cleavage of lignin β-O-4 aryl ether bonds via net internal    hydrogen transfer. Green Chemistry 2013, 15:1373-1381.-   Reiter J, Pick A, Wiemann L O, Schieder D, Sieber V: A novel natural    NADH and NADPH dependent glutathione reductase as tool in    biotechnological applications. JSM Biotechnol Bioeng 2014,    2:1028-1035.-   Rosini E, Allegretti C, Melis R, Cerioli L, Conti G, Pollegioni L,    D'Arrigo P: Cascade enzymatic cleavage of the β-O-4 linkage in a    lignin model compound. Catalysis Science & Technology 2016,    6:2195-2205.-   Santos R B, Hart P, Jameel H, Chang H. Wood based lignin reactions    important to the biorefinery and pulp and paper industries.    BioResources 2013, 8(1):1456-1477.-   Sato Y, et al. (2009) Identification of three alcohol dehydrogenase    genes involved in the stereospecific catabolism of    arylglycerol-β-aryl ether by Sphingobium sp. strain SYK-6. Appl    Environ Microbiol 75(16):5195-5201.-   Schäfer, A., Tauch, A., Jager, W., Kalinowski, J., Thierbach, G.,    and Pühler, A. (1994). Small mobilizable multi-purpose cloning    vectors derived from the Escherichia coli plasmids pK18 and pK19:    selection of defined deletions in the chromosome of Corynebacterium    glutamicum. Gene 145, 69-73.-   Shevchuk N A, Bryksin A V, Nusinovich Y A, Cabello F C, Sutherland    M, Ladisch S: Construction of long DNA molecules using long    PCR-based fusion of several fragments simultaneously. Nucleic Acids    Research 2004, 32.-   Shuai L, Amiri M T, Questell-Santiago Y M, Héroguel F, Li Y, Kim H,    Meilan R, Chapple C, Ralph J, Luterbacher J S: Stabilization with    formaldehyde facilitates the high-yield production of monomers from    lignin during integrated biomass depolymerization. Science 2016,    354(6310):329-333.-   Simon, R., Priefer, U., and Pühler, A. (1983). A Broad Host Range    Mobilization System for In Vivo Genetic Engineering: Transposon    Mutagenesis in Gram Negative Bacteria. Nat Biotech 1, 784-791.-   Sinha A K, Sharma U K, Sharma N: A comprehensive review on vanilla    flavor: Extraction, isolation and quantification of vanillin and    others constituents. International Journal of Food Sciences and    Nutrition 2008, 59:299-326.-   Sistrom W R (1962) The kinetics of the synthesis of photopigments in    Rhodopseudomonas spheroides. J Gen Microbiol 28:607-616.-   Stanier R Y, Palleroni N J, Doudoroff M (1966) The aerobic    pseudomonads: a taxonomic study. J Gen Microbiol 43(2):159-271.-   Stewart J J, Akiyama T, Chapple C, Ralph J, Mansfield S D: The    effects on lignin structure of overexpression of ferulate    5-hydroxylase in hybrid poplar. Plant Physiology 2009, 150:621-635.-   Stolz A, et al. (2000) Description of Sphingomonas xenophaga sp.    nov. for strains BN6^(T) and-   N,N which degrade xenobiotic aromatic compounds. Int J Syst Evol    Microbiol 50 Pt 1:35-41.-   Stourman N V, et al. (2011) Structure and function of YghU, a    nu-class glutathione transferase related to YfcG from Escherichia    coli. Biochemistry 50(7):1274-1281.-   Studier F W (2005) Protein production by auto-induction in high    density shaking cultures. Protein Expr Purif 41(1):207-234.-   Sugimoto T, Akiyama T, Matsumoto Y, Meshitsuka G: The erythro/threo    ratio of β-O-4 structures as an important structural characteristic    of lignin—Part 2. Changes in erythro/threo (E/T) ratio of β-O-4    structures during delignification reactions. Holzforschung 2002,    56:416-421.-   Tanamura K, Kasai D, Nakamura M, Katayama Y, Fukuda M, Masai E:    Identification of the third glutathione S-transferase gene involved    in the stereospecific cleavage of β-aryl ether in Sphingobium sp.    strain SYK-6. Journal of Biotechnology 2010, 150:S235-S235.-   Tavano C L, Podevels A M, Donohue T J (2005) Identification of genes    required for recycling reducing power during photosynthetic growth.    J Bacteriol 187(15):5249-5258.-   Taylor, R. G., Walker, D. C., and McInnes, R. R. (1993). E. coli    host strains significantly affect the quality of small scale plasmid    DNA preparations used for sequencing. Nucleic Acids Res. 21,    1677-1678-   Thuillier, A., Roret, T., Favier, F., Gelhaye, E., Jacquot, J.-P.,    Didierjean, C., and Morel-Rouhier, M. (2013). Atypical features of a    Ure2p glutathione transferase from Phanerochaete chrysosporium. FEBS    Lett. 587, 2125-2130.-   Tsien R Y. (1998) The green fluorescent protein. Annu Rev Biochem.    67:509-44.-   U.S. DOE (2015) Lignocellulose Biomass for Advanced Biofuels and    Bioproducts: Workshop Report, DOE/SC-0170. U.S. Department of Energy    Office of Science. Available at:    http://genomicscience.energy.gov/biofuels/lignocellulose/[Accessed    May 17, 2017].-   Vicuña R, González B, Mozuch M D, Kirk T K (1987) Metabolism of    lignin model compounds of the arylglycerol-β-aryl ether type by    Pseudomonas acidovorans D(3). Appl Environ Microbiol    53(11):2605-2609.-   Wadington M C, Ladner J E, Stourman N V, Harp J M, Armstrong R    N (2009) Analysis of the structure and function of YfcG from    Escherichia coli reveals an efficient and unique disulfide bond    reductase. Biochemistry 48(28):6559-6561.-   Wadington M C, Ladner J E, Stourman N V, Harp J M, Armstrong R    N (2010) Correction to Analysis of the structure and function of    YfcG from Escherichia coli reveals an efficient and unique disulfide    bond reductase. Biochemistry 49(50):10765.-   Wood W B (1966) Host specificity of DNA produced by Escherichia    coli: bacterial mutations affecting the restriction and modification    of DNA. J Mol Biol 16(1):118-133.

EXEMPLARY VERSIONS OF THE INVENTION

Various exemplary versions of the invention are as follows.

Version 1: A method of processing lignin, comprising contacting lignincomprising β-O-4 ether linkages in vitro with:

-   -   a dehydrogenase comprising at least one of LigD, LigO, LigN, and        LigL;    -   a β-etherase comprising at least one of LigE, LigF, LigP, and an        enzyme comprising a first polypeptide having an amino acid        sequence of SEQ ID NO:40 or an amino acid sequence at least        about 95% identical thereto and a second polypeptide having an        amino acid sequence of SEQ ID NO:42 or an amino acid sequence at        least about 95% identical thereto; and    -   a glutathione lyase comprising any one or more of LigG and a        non-stereospecific glutathione lyase comprising an amino acid        sequence at least about 80%, 85%, 90%, or 95% identical to any        of:    -   SEQ ID NO:18 (NaGST_(Nu));    -   residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));    -   SEQ ID NO:22 (SYK6GST_(Nu));    -   residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu)); SEQ        ID NO:26 (ecYghU);    -   residues 21-313 of SEQ ID NO:28 (recombinant ecYghU);    -   SEQ ID NO:30 (ecYfcG);    -   SEQ ID NO:32 (ssYghU);    -   SEQ ID NO:34 (GST3); and    -   SEQ ID NO:36 (PcUre2pB1).

Version 2. The method of version 1, wherein the glutathione lyasecomprises the non-stereospecific glutathione lyase.

Version 3. The method of version 1, wherein the glutathione lyasecomprises the non-stereospecific glutathione lyase and thenon-stereospecific glutathione lyase comprises an amino acid sequence atleast about 80%, 85%, 90%, or 95% identical to any of:

-   -   SEQ ID NO:18 (NaGST_(Nu));    -   residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));    -   SEQ ID NO:22 (SYK6GST_(Nu));    -   residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu));    -   SEQ ID NO:26 (ecYghU);    -   residues 21-313 of SEQ ID NO:28 (recombinant ecYghU);    -   SEQ ID NO:30 (ecYfcG);    -   SEQ ID NO:32 (ssYghU);    -   SEQ ID NO:34 (GST3); and    -   SEQ ID NO:36 (PcUre2pB1).

Version 4. The method of version 1, wherein the glutathione lyasecomprises the non-stereospecific glutathione lyase and thenon-stereospecific glutathione lyase comprises an amino acid sequence atleast about 80%, 85%, 90%, or 95% identical to any of:

-   -   SEQ ID NO:18 (NaGST_(Nu));    -   residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));    -   SEQ ID NO:22 (SYK6GST_(Nu));    -   residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu));    -   SEQ ID NO:26 (ecYghU); and    -   residues 21-313 of SEQ ID NO:28 (recombinant ecYghU).

Version 5. The method of version 1, wherein the glutathione lyasecomprises the non-stereospecific glutathione lyase and thenon-stereospecific glutathione lyase comprises an amino acid sequence atleast about 90% or 95% identical to any of:

-   -   SEQ ID NO:18 (NaGST_(Nu));    -   residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));    -   SEQ ID NO:22 (SYK6GST_(Nu));    -   residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu));    -   SEQ ID NO:26 (ecYghU); and    -   residues 21-313 of SEQ ID NO:28 (recombinant ecYghU).

Version 6. The method of any one of versions 1-5, wherein thenon-stereospecific glutathione lyase comprises at least one, at leasttwo, at least three, at least four, at least five, at least six, atleast seven, or all of:

-   -   threonine or a conservative variant of threonine at a position        corresponding to position 51 of SEQ ID NO:18 (NaGST_(Nu));    -   asparagine or a conservative variant of asparagine at a position        corresponding to position 53 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamine or a conservative variant of glutamine at a position        corresponding to position 86 of SEQ ID NO:18 (NaGST_(Nu));    -   lysine, a conservative variant of lysine, arginine, or a        conservative variant of arginine at a position corresponding to        position 99 of SEQ ID NO:18 (NaGST_(Nu));    -   isoleucine or a conservative variant of isoleucine at a position        corresponding to position 100 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamate or a conservative variant of glutamate at a position        corresponding to position 116 of SEQ ID NO:18 (NaGST_(Nu));    -   serine, threonine, a conservative variant of serine, or a        conservative variant of threonine at a position corresponding to        position 117 of SEQ ID NO:18 (NaGST_(Nu));    -   arginine or a conservative variant of arginine at a position        corresponding to position 177 of SEQ ID NO:18 (NaGST_(Nu)).

Version 7. The method of any one of versions 1-5, wherein thenon-stereospecific glutathione lyase comprises at least one, at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least nine, at least ten, or all of:

-   -   asparagine or a conservative variant of asparagine at a position        corresponding to position 25 of SEQ ID NO:18 (NaGST_(Nu));    -   threonine or a conservative variant of threonine at a position        corresponding to position 51 of SEQ ID NO:18 (NaGST_(Nu));    -   asparagine or a conservative variant of asparagine at a position        corresponding to position 53 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamine or a conservative variant of glutamine at a position        corresponding to position 86 of SEQ ID NO:18 (NaGST_(Nu));    -   lysine, a conservative variant of lysine, arginine, or a        conservative variant of arginine at a position corresponding to        position 99 of SEQ ID NO:18 (NaGST_(Nu));    -   isoleucine or a conservative variant of isoleucine at a position        corresponding to position 100 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamate or a conservative variant of glutamate at a position        corresponding to position 116 of SEQ ID NO:18 (NaGST_(Nu));    -   serine, threonine, a conservative variant of serine, or a        conservative variant of threonine at a position corresponding to        position 117 of SEQ ID NO:18 (NaGST_(Nu));    -   tyrosine or a conservative variant of tyrosine at a position        corresponding to position 166 of SEQ ID NO:18 (NaGST_(Nu));    -   arginine or a conservative variant of arginine at a position        corresponding to position 177 of SEQ ID NO:18 (NaGST_(Nu)); and    -   tyrosine or a conservative variant of tyrosine at a position        corresponding to position 224 of SEQ ID NO:18 (NaGST_(Nu)).

Version 8. The method of any one of versions 1-8, wherein the contactingoccurs in the presence of a glutathione (GSH) reductase that catalyzesreduction of glutathione disulfide (GSSG).

Version 9. The method of version 8, wherein the GSH reductase comprisesan amino acid sequence at least about 95% identical to SEQ ID NO:38(AvGR).

Version 10. The method of any one of versions 1-9, wherein thecontacting releases at least one of a monomeric phenylpropanoid unit anda monomeric flavone.

Version 11. The method of any one of versions 1-10, wherein thecontacting releases at least one of a monomeric guaiacyl phenylpropanoidunit, a monomeric syringyl phenylpropanoid unit, a monomericp-hydroxyphenyl phenylpropanoid unit, and a monomeric tricin unit.

Version 12. The method of any one of versions 1-11, wherein the lignincomprises an average molecular weight (MW) of from about 600 to about20,000.

Version 13. A composition, comprising:

-   -   lignin comprising β-O-4 ether linkages;    -   a dehydrogenase comprising at least one of LigD, LigO, LigN, and        LigL;    -   a β-etherase comprising at least one of LigE, LigF, LigP, and an        enzyme comprising a first polypeptide having an amino acid        sequence of SEQ ID NO:40 or an amino acid sequence at least        about 95% identical thereto and a second polypeptide having an        amino acid sequence of SEQ ID NO:42 or an amino acid sequence at        least about 95% identical thereto; and    -   a glutathione lyase comprising any one or more of LigG and a        non-stereospecific glutathione lyase comprising an amino acid        sequence at least about 80%, 85%, 90%, or 95% identical to any        of:    -   SEQ ID NO:18 (NaGST_(Nu));    -   residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));    -   SEQ ID NO:22 (SYK6GST_(Nu));    -   residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu));    -   SEQ ID NO:26 (ecYghU);    -   residues 21-313 of SEQ ID NO:28 (recombinant ecYghU);    -   SEQ ID NO:30 (ecYfcG);    -   SEQ ID NO:32 (ssYghU);    -   SEQ ID NO:34 (GST3); and    -   SEQ ID NO:36 (PcUre2pB1).

Version 14. The composition of version 13, wherein the glutathione lyasecomprises the non-stereospecific glutathione lyase.

Version 15. The composition of version 13, wherein the glutathione lyasecomprises the non-stereospecific glutathione lyase and thenon-stereospecific glutathione lyase comprises an amino acid sequence atleast about 80%, 85%, 90%, or 95% identical to any of:

-   -   SEQ ID NO:18 (NaGST_(Nu));    -   residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));    -   SEQ ID NO:22 (SYK6GST_(Nu));    -   residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu));    -   SEQ ID NO:26 (ecYghU);    -   residues 21-313 of SEQ ID NO:28 (recombinant ecYghU);    -   SEQ ID NO:30 (ecYfcG);    -   SEQ ID NO:32 (ssYghU);    -   SEQ ID NO:34 (GST3); and    -   SEQ ID NO:36 (PcUre2pB1).

Version 16. The composition of version 13, wherein the glutathione lyasecomprises the non-stereospecific glutathione lyase and thenon-stereospecific glutathione lyase comprises an amino acid sequence atleast about 80%, 85%, 90%, or 95% identical to any of:

-   -   SEQ ID NO:18 (NaGST_(Nu));    -   residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));    -   SEQ ID NO:22 (SYK6GST_(Nu));    -   residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu));    -   SEQ ID NO:26 (ecYghU); and    -   residues 21-313 of SEQ ID NO:28 (recombinant ecYghU).

Version 17. The composition of version 13, wherein the glutathione lyasecomprises the non-stereospecific glutathione lyase and thenon-stereospecific glutathione lyase comprises an amino acid sequence atleast about 90% or 95% identical to any of:

-   -   SEQ ID NO:18 (NaGST_(Nu));    -   residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));    -   SEQ ID NO:22 (SYK6GST_(Nu));    -   residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu));    -   SEQ ID NO:26 (ecYghU); and    -   residues 21-313 of SEQ ID NO:28 (recombinant ecYghU).

Version 18. The composition of any one of versions 13-17, wherein thenon-stereospecific glutathione lyase comprises at least one, at leasttwo, at least three, at least four, at least five, at least six, atleast seven, or all of:

-   -   threonine or a conservative variant of threonine at a position        corresponding to position 51 of SEQ ID NO:18 (NaGST_(Nu));    -   asparagine or a conservative variant of asparagine at a position        corresponding to position 53 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamine or a conservative variant of glutamine at a position        corresponding to position 86 of SEQ ID NO:18 (NaGST_(Nu));    -   lysine, a conservative variant of lysine, arginine, or a        conservative variant of arginine at a position corresponding to        position 99 of SEQ ID NO:18 (NaGST_(Nu));    -   isoleucine or a conservative variant of isoleucine at a position        corresponding to position 100 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamate or a conservative variant of glutamate at a position        corresponding to position 116 of SEQ ID NO:18 (NaGST_(Nu));    -   serine, threonine, a conservative variant of serine, or a        conservative variant of threonine at a position corresponding to        position 117 of SEQ ID NO:18 (NaGST_(Nu));    -   arginine or a conservative variant of arginine at a position        corresponding to position 177 of SEQ ID NO:18 (NaGST_(Nu)).

Version 19. The composition of any one of versions 13-17, wherein thenon-stereospecific glutathione lyase comprises at least one, at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least nine, at least ten, or all of:

-   -   asparagine or a conservative variant of asparagine at a position        corresponding to position 25 of SEQ ID NO:18 (NaGST_(Nu));    -   threonine or a conservative variant of threonine at a position        corresponding to position 51 of SEQ ID NO:18 (NaGST_(Nu));    -   asparagine or a conservative variant of asparagine at a position        corresponding to position 53 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamine or a conservative variant of glutamine at a position        corresponding to position 86 of SEQ ID NO:18 (NaGST_(Nu));    -   lysine, a conservative variant of lysine, arginine, or a        conservative variant of arginine at a position corresponding to        position 99 of SEQ ID NO:18 (NaGST_(Nu));    -   isoleucine or a conservative variant of isoleucine at a position        corresponding to position 100 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamate or a conservative variant of glutamate at a position        corresponding to position 116 of SEQ ID NO:18 (NaGST_(Nu));    -   serine, threonine, a conservative variant of serine, or a        conservative variant of threonine at a position corresponding to        position 117 of SEQ ID NO:18 (NaGST_(Nu));    -   tyrosine or a conservative variant of tyrosine at a position        corresponding to position 166 of SEQ ID NO:18 (NaGST_(Nu));    -   arginine or a conservative variant of arginine at a position        corresponding to position 177 of SEQ ID NO:18 (NaGST_(Nu)); and    -   tyrosine or a conservative variant of tyrosine at a position        corresponding to position 224 of SEQ ID NO:18 (NaGST_(Nu)).

Version 20. The composition of any one of versions 13-19, furthercomprising a glutathione (GSH) reductase that catalyzes reduction ofglutathione disulfide (GSSG).

Version 21. The composition of version 20, wherein the GSH reductasecomprises an amino acid sequence at least about 95% identical to SEQ IDNO:38 (AvGR).

Version 22. A method of chemical conversion, comprising contacting afirst compound in vitro with a non-stereospecific glutathione lyase toyield a second compound, wherein:

-   -   the non-stereospecific glutathione lyase comprises an amino acid        sequence at least about 80%, 85%, 90%, or 95% identical to any        of:        -   SEQ ID NO:18 (NaGST_(Nu));        -   residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));        -   SEQ ID NO:22 (SYK6GST_(Nu));        -   residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu));        -   SEQ ID NO:26 (ecYghU);        -   residues 21-313 of SEQ ID NO:28 (recombinant ecYghU);        -   SEQ ID NO:30 (ecYfcG);        -   SEQ ID NO:32 (ssYghU);        -   SEQ ID NO:34 (GST3); and        -   SEQ ID NO:36 (PcUre2pB1)    -   the first compound has a structure of Formula I or a salt        thereof:

wherein:

-   -   R¹, R², and R³ are each independently —H, —OH, —O-alkyl,        —O-lignin, or -lignin;    -   R⁴ is —H, —OH, —SH, —COOH, —SO₃H, or —O-lignin; and    -   SG is glutathione bound in an S or R configuration; and    -   the second compound has a structure of Formula II or a salt        thereof:

wherein R¹, R², R³, and R⁴ are as defined above.

-   -   Version 23. The method of version 22, wherein:        -   R¹ in Formula I and Formula II is —H or —OCH₃;        -   R² in Formula I and Formula II is —OH;        -   R³ in Formula I and Formula II is —H or —OCH₃; and        -   R⁴ in Formula I and Formula II is —OH.

Version 24. The method of any one of versions 22-23, wherein thenon-stereospecific glutathione lyase comprises an amino acid sequence atleast about 80%, 85%, 90%, or 95% identical to any of:

-   -   SEQ ID NO:18 (NaGST_(Nu));    -   residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));    -   SEQ ID NO:22 (SYK6GST_(Nu));    -   residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu));    -   SEQ ID NO:26 (ecYghU); and    -   residues 21-313 of SEQ ID NO:28 (recombinant ecYghU).

Version 25. The method of any one of versions 22-23, wherein thenon-stereospecific glutathione lyase comprises an amino acid sequence atleast about 90% or 95% identical to any of:

-   -   SEQ ID NO:18 (NaGST_(Nu));    -   residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));    -   SEQ ID NO:22 (SYK6GST_(Nu));    -   residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu));    -   SEQ ID NO:26 (ecYghU); and    -   residues 21-313 of SEQ ID NO:28 (recombinant ecYghU).

Version 26. The method of any one of versions 22-25, wherein thenon-stereospecific glutathione lyase comprises at least one, at leasttwo, at least three, at least four, at least five, at least six, atleast seven, or all of:

-   -   threonine or a conservative variant of threonine at a position        corresponding to position 51 of SEQ ID NO:18 (NaGST_(Nu));    -   asparagine or a conservative variant of asparagine at a position        corresponding to position 53 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamine or a conservative variant of glutamine at a position        corresponding to position 86 of SEQ ID NO:18 (NaGST_(Nu));    -   lysine, a conservative variant of lysine, arginine, or a        conservative variant of arginine at a position corresponding to        position 99 of SEQ ID NO:18 (NaGST_(Nu));    -   isoleucine or a conservative variant of isoleucine at a position        corresponding to position 100 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamate or a conservative variant of glutamate at a position        corresponding to position 116 of SEQ ID NO:18 (NaGST_(Nu));    -   serine, threonine, a conservative variant of serine, or a        conservative variant of threonine at a position corresponding to        position 117 of SEQ ID NO:18 (NaGST_(Nu));    -   arginine or a conservative variant of arginine at a position        corresponding to position 177 of SEQ ID NO:18 (NaGST_(Nu)).

Version 27. The method of any one of versions 22-25, wherein thenon-stereospecific glutathione lyase comprises at least one, at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least nine, at least ten, or all of:

-   -   asparagine or a conservative variant of asparagine at a position        corresponding to position 25 of SEQ ID NO:18 (NaGST_(Nu));    -   threonine or a conservative variant of threonine at a position        corresponding to position 51 of SEQ ID NO:18 (NaGST_(Nu));    -   asparagine or a conservative variant of asparagine at a position        corresponding to position 53 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamine or a conservative variant of glutamine at a position        corresponding to position 86 of SEQ ID NO:18 (NaGST_(Nu));    -   lysine, a conservative variant of lysine, arginine, or a        conservative variant of arginine at a position corresponding to        position 99 of SEQ ID NO:18 (NaGST_(Nu));    -   isoleucine or a conservative variant of isoleucine at a position        corresponding to position 100 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamate or a conservative variant of glutamate at a position        corresponding to position 116 of SEQ ID NO:18 (NaGST_(Nu));    -   serine, threonine, a conservative variant of serine, or a        conservative variant of threonine at a position corresponding to        position 117 of SEQ ID NO:18 (NaGST_(Nu));    -   tyrosine or a conservative variant of tyrosine at a position        corresponding to position 166 of SEQ ID NO:18 (NaGST_(Nu));    -   arginine or a conservative variant of arginine at a position        corresponding to position 177 of SEQ ID NO:18 (NaGST_(Nu)); and    -   tyrosine or a conservative variant of tyrosine at a position        corresponding to position 224 of SEQ ID NO:18 (NaGST_(Nu)).

Version 28. The method of any of versions 22-27, wherein the contactingoccurs in the presence of a glutathione (GSH) reductase that catalyzesreduction of glutathione disulfide (GSSG).

Version 29. The method of version 28, wherein the GSH reductasecomprises an amino acid sequence at least about 95% identical to SEQ IDNO:38 (AvGR).

Version 30. The method of any one of versions 22-29, further comprisingcontacting lignin comprising β-O-4 ether linkages in vitro with enzymesto generate the first compound, wherein the enzymes comprise:

-   -   a dehydrogenase comprising at least one of LigD, LigO, LigN, and        LigL; and    -   a β-etherase comprising at least one of LigE, LigF, LigP, and an        enzyme comprising a first polypeptide having an amino acid        sequence of SEQ ID NO:40 or an amino acid sequence at least        about 95% identical thereto and    -   a second polypeptide having an amino acid sequence of SEQ ID        NO:42 or an amino acid sequence at least about 95% identical        thereto.

Version 31. A recombinant non-stereospecific glutathione lyasecomprising an amino acid sequence at least about 80%, 85%, 90%, or 95%identical to any of:

-   -   SEQ ID NO:18 (NaGST_(Nu));    -   residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));    -   SEQ ID NO:22 (SYK6GST_(Nu)); and    -   residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu)).

Version 32. The glutathione lyase of version 31, comprising an aminoacid sequence at least about 80%, 85%, 90%, or 95% identical to any of:

-   -   SEQ ID NO:18 (NaGST_(Nu));    -   residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));    -   SEQ ID NO:22 (SYK6GST_(Nu)); and    -   residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu)).

Version 33. The glutathione lyase of version 31, comprising an aminoacid sequence at least about 90% or 95% identical to any of:

-   -   SEQ ID NO:18 (NaGST_(Nu));    -   residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));    -   SEQ ID NO:22 (SYK6GST_(Nu)); and    -   residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST_(Nu)).

Version 34. The glutathione lyase of any one of versions 31-33, whereinthe non-stereospecific glutathione lyase comprises at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, or all of:

-   -   threonine or a conservative variant of threonine at a position        corresponding to position 51 of SEQ ID NO:18 (NaGST_(Nu));    -   asparagine or a conservative variant of asparagine at a position        corresponding to position 53 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamine or a conservative variant of glutamine at a position        corresponding to position 86 of SEQ ID NO:18 (NaGST_(Nu));    -   lysine, a conservative variant of lysine, arginine, or a        conservative variant of arginine at a position corresponding to        position 99 of SEQ ID NO:18 (NaGST_(Nu));    -   isoleucine or a conservative variant of isoleucine at a position        corresponding to position 100 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamate or a conservative variant of glutamate at a position        corresponding to position 116 of SEQ ID NO:18 (NaGST_(Nu));    -   serine, threonine, a conservative variant of serine, or a        conservative variant of threonine at a position corresponding to        position 117 of SEQ ID NO:18 (NaGST_(Nu));    -   arginine or a conservative variant of arginine at a position        corresponding to position 177 of SEQ ID NO:18 (NaGST_(Nu)).

Version 35. The glutathione lyase of any one of versions 31-33, whereinthe non-stereospecific glutathione lyase comprises at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, at least ten, or all of:

-   -   asparagine or a conservative variant of asparagine at a position        corresponding to position 25 of SEQ ID NO:18 (NaGST_(Nu));    -   threonine or a conservative variant of threonine at a position        corresponding to position 51 of SEQ ID NO:18 (NaGST_(Nu));    -   asparagine or a conservative variant of asparagine at a position        corresponding to position 53 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamine or a conservative variant of glutamine at a position        corresponding to position 86 of SEQ ID NO:18 (NaGST_(Nu));    -   lysine, a conservative variant of lysine, arginine, or a        conservative variant of arginine at a position corresponding to        position 99 of SEQ ID NO:18 (NaGST_(Nu));    -   isoleucine or a conservative variant of isoleucine at a position        corresponding to position 100 of SEQ ID NO:18 (NaGST_(Nu));    -   glutamate or a conservative variant of glutamate at a position        corresponding to position 116 of SEQ ID NO:18 (NaGST_(Nu));    -   serine, threonine, a conservative variant of serine, or a        conservative variant of threonine at a position corresponding to        position 117 of SEQ ID NO:18 (NaGST_(Nu));    -   tyrosine or a conservative variant of tyrosine at a position        corresponding to position 166 of SEQ ID NO:18 (NaGST_(Nu));    -   arginine or a conservative variant of arginine at a position        corresponding to position 177 of SEQ ID NO:18 (NaGST_(Nu)); and    -   tyrosine or a conservative variant of tyrosine at a position        corresponding to position 224 of SEQ ID NO:18 (NaGST_(Nu)).

Version 36. The glutathione lyase of any one of versions 31-35, whereinthe glutathione lyase comprises at least one non-native modificationselected from the group consisting of an amino acid addition, an aminoacid deletion, and an amino acid substitution.

We claim:
 1. A method of processing lignin, comprising contacting lignincomprising β-O-4 ether linkages in vitro with: a dehydrogenasecomprising at least one of LigD, LigO, LigN, and LigL; a β-etherasecomprising at least one of LigE, LigF, LigP, and an enzyme comprising afirst polypeptide having an amino acid sequence of SEQ ID NO:40 or anamino acid sequence at least about 95% identical thereto and a secondpolypeptide having an amino acid sequence of SEQ ID NO:42 or an aminoacid sequence at least about 95% identical thereto; and anon-stereospecific glutathione lyase comprising an amino acid sequenceat least about 80% identical to any of: SEQ ID NO:18 (NaGST_(Nu));residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu)); SEQ ID NO:22(SYK6GST_(Nu)); residues 21-324 of SEQ ID NO:24 (recombinantSYK6GST_(Nu)); SEQ ID NO:26 (ecYghU); residues 21-313 of SEQ ID NO:28(recombinant ecYghU); and SEQ ID NO:32 (ssYghU), wherein thenon-stereospecific glutathione lyase comprises at least four of:threonine or a conservative variant of threonine at a positioncorresponding to position 51 of SEQ ID NO:18 (NaGST_(Nu)); asparagine ora conservative variant of asparagine at a position corresponding toposition 53 of SEQ ID NO:18 (NaGST_(Nu)); glutamine or a conservativevariant of glutamine at a position corresponding to position 86 of SEQID NO:18 (NaGST_(Nu)); lysine, a conservative variant of lysine,arginine, or a conservative variant of arginine at a positioncorresponding to position 99 of SEQ ID NO:18 (NaGST_(Nu)); isoleucine ora conservative variant of isoleucine at a position corresponding toposition 100 of SEQ ID NO:18 (NaGST_(Nu)); glutamate or a conservativevariant of glutamate at a position corresponding to position 116 of SEQID NO:18 (NaGST_(Nu)); serine, threonine, a conservative variant ofserine, or a conservative variant of threonine at a positioncorresponding to position 117 of SEQ ID NO:18 (NaGST_(Nu)); and arginineor a conservative variant of arginine at a position corresponding toposition 177 of SEQ ID NO:18 (NaGST_(Nu)).
 2. The method of claim 1,wherein the non-stereospecific glutathione lyase comprises an amino acidsequence at least about 85% identical to any of: SEQ ID NO:18(NaGST_(Nu)); residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));SEQ ID NO:22 (SYK6GST_(Nu)); residues 21-324 of SEQ ID NO:24(recombinant SYK6GST_(Nu)); SEQ ID NO:26 (ecYghU); residues 21-313 ofSEQ ID NO:28 (recombinant ecYghU); and SEQ ID NO:32 (ssYghU).
 3. Themethod of claim 1, wherein the non-stereospecific glutathione lyasecomprises an amino acid sequence at least about 90% identical to any of:SEQ ID NO:18 (NaGST_(Nu)); residues 21-313 of SEQ ID NO:20 (recombinantNaGST_(Nu)); SEQ ID NO:22 (SYK6GST_(Nu)); residues 21-324 of SEQ IDNO:24 (recombinant SYK6GST_(Nu)); SEQ ID NO:26 (ecYghU); and residues21-313 of SEQ ID NO:28 (recombinant ecYghU).
 4. The method of claim 1,wherein the non-stereospecific glutathione lyase comprises an amino acidsequence at least about 95% identical to any of: SEQ ID NO:18(NaGST_(Nu)); residues 21-313 of SEQ ID NO:20 (recombinant NaGST_(Nu));SEQ ID NO:22 (SYK6GST_(Nu)); residues 21-324 of SEQ ID NO:24(recombinant SYK6GST_(Nu)); SEQ ID NO:26 (ecYghU); and residues 21-313of SEQ ID NO:28 (recombinant ecYghU).
 5. The method of claim 1, whereinthe non-stereospecific glutathione lyase comprises at least seven of:asparagine or a conservative variant of asparagine at a positioncorresponding to position 25 of SEQ ID NO:18 (NaGST_(Nu)); threonine ora conservative variant of threonine at a position corresponding toposition 51 of SEQ ID NO:18 (NaGST_(Nu)); asparagine or a conservativevariant of asparagine at a position corresponding to position 53 of SEQID NO:18 (NaGST_(Nu)); glutamine or a conservative variant of glutamineat a position corresponding to position 86 of SEQ ID NO:18 (NaGST_(Nu));lysine, a conservative variant of lysine, arginine, or a conservativevariant of arginine at a position corresponding to position 99 of SEQ IDNO:18 (NaGST_(Nu)); isoleucine or a conservative variant of isoleucineat a position corresponding to position 100 of SEQ ID NO:18(NaGST_(Nu)); glutamate or a conservative variant of glutamate at aposition corresponding to position 116 of SEQ ID NO:18 (NaGST_(Nu));serine, threonine, a conservative variant of serine, or a conservativevariant of threonine at a position corresponding to position 117 of SEQID NO:18 (NaGST_(Nu)); tyrosine or a conservative variant of tyrosine ata position corresponding to position 166 of SEQ ID NO:18 (NaGST_(Nu));arginine or a conservative variant of arginine at a positioncorresponding to position 177 of SEQ ID NO:18 (NaGST_(Nu)); and tyrosineor a conservative variant of tyrosine at a position corresponding toposition 224 of SEQ ID NO:18 (NaGST_(Nu)).
 6. The method of claim 1,wherein the contacting occurs in the presence of a glutathione (GSH)reductase that catalyzes reduction of glutathione disulfide (GSSG). 7.The method of claim 6, wherein the GSH reductase comprises an amino acidsequence at least about 95% identical to SEQ ID NO:38 (AvGR).
 8. Themethod of claim 1, wherein the contacting releases at least one of amonomeric phenylpropanoid unit and a monomeric flavone.
 9. The method ofclaim 1, wherein the contacting releases at least one of a monomericguaiacyl phenylpropanoid unit, a monomeric syringyl phenylpropanoidunit, a monomeric p-hydroxyphenyl phenylpropanoid unit, and a monomerictricin unit.
 10. The method of claim 1, wherein the lignin comprises anaverage molecular weight (MW) of from about 600 to about 20,000.
 11. Themethod of claim 1, wherein the dehydrogenase comprises at least one ofLigD and LigO and at least one of LigL and LigN.
 12. The method of claim1, wherein the dehydrogenase comprises LigD and LigN.
 13. The method ofclaim 1, wherein the β-etherase comprises LigF and at least one of LigE,LigP, and the enzyme comprising the first polypeptide having the aminoacid sequence of SEQ ID NO:40 or the amino acid sequence at least about95% identical thereto and the second polypeptide having the amino acidsequence of SEQ ID NO:42 or the amino acid sequence at least about 95%identical thereto.
 14. The method of claim 1, wherein the β-etherasecomprises LigF and LigE.
 15. The method of claim 1, wherein thenon-stereospecific glutathione lyase comprises an amino acid sequence atleast about 95% identical to SEQ ID NO:18 (NaGST_(Nu)).
 16. The methodof claim 1, wherein: the dehydrogenase comprises at least one of LigDand LigO and at least one of LigL and LigN; the β-etherase comprisesLigF and at least one of LigE, LigP, and the enzyme comprising the firstpolypeptide having the amino acid sequence of SEQ ID NO:40 or the aminoacid sequence at least about 95% identical thereto and the secondpolypeptide having the amino acid sequence of SEQ ID NO:42 or the aminoacid sequence at least about 95% identical thereto; thenon-stereospecific glutathione lyase comprises an amino acid sequence atleast about 95% identical to SEQ ID NO:18 (NaGST_(Nu)); and thecontacting occurs in the presence of a glutathione (GSH) reductase thatcatalyzes reduction of glutathione disulfide (GSSG) and comprises anamino acid sequence at least about 95% identical to SEQ ID NO:38 (AvGR).17. The method of claim 1, wherein: the dehydrogenase comprises LigD andLigN; the β-etherase comprises LigF and LigE; the non-stereospecificglutathione lyase comprises an amino acid sequence at least about 95%identical to SEQ ID NO:18 (NaGST_(Nu)); and the contacting occurs in thepresence of a glutathione (GSH) reductase that catalyzes reduction ofglutathione disulfide (GSSG) and comprises an amino acid sequence atleast about 95% identical to SEQ ID NO:38 (AvGR).